Optimized factor viii gene

ABSTRACT

The present invention also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject a codon optimized Factor VIII nucleic acid sequence or the polypeptide encoded thereby.

BACKGROUND OF THE INVENTION

The blood coagulation pathway, in part, involves the formation of anenzymatic complex of Factor VIIIa (FVIIIa) and Factor IXa (FIXa) (Xasecomplex) on the surface of platelets. FIXa is a serine protease withrelatively weak catalytic activity without its cofactor FVIIIa. The Xasecomplex cleaves Factor X (FX) into Factor Xa (FXa), which in turninteracts with Factor Va (FVa) to cleave prothrombin and generatethrombin. Hemophilia A is a bleeding disorder caused by mutations and/ordeletions in the FVIII (FVIII) gene resulting in a deficiency of FVIIIactivity (Peyvandi et al. 2006). In some cases, patients have reducedlevels of FVIII due to the presence of FVIII inhibitors, such asanti-FVIII antibodies.

Hemophilia A is characterized by spontaneous hemorrhage and excessivebleeding. Over time, the repeated bleeding into muscles and joints,which often begins in early childhood, results in hemophilic arthropathyand irreversible joint damage. This damage is progressive and can leadto severely limited mobility of joints, muscle atrophy and chronic pain(Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96 (2003), whichis herein incorporated by reference in its entirety).

The disease can be treated by replacement therapy targeting restorationof FVIII activity to 1 to 5% of normal levels to prevent spontaneousbleeding (see, e.g., Mannucci, P. M., et al., N. Engl. J Med. 344:1773-9(2001), herein incorporated by reference in its entirety). There areplasma-derived and recombinant FVIII products available to treatbleeding episodes on-demand or to prevent bleeding episodes fromoccurring by treating prophylactically. Based on the half-life of theseproducts (10-12 hr) (White G. C., et al., Thromb. Haemost. 77:660-7(1997); Morfini, M., Haemophilia 9 (suppl 1): 94-99; discussion 100(2003)), treatment regimens require frequent intravenous administration,commonly two to three times weekly for prophylaxis and one to threetimes daily for on-demand treatment (Manco-Johnson, M. J., et al., N.Engl. J. Med. 357:535-544 (2007)), each of which is incorporated hereinby reference in its entirety. Such frequent administration isinconvenient and costly.

A major impediment in providing a low-cost recombinant FVIII protein topatients is the high cost of commercial production. FVIII proteinexpresses poorly in heterologous expression systems, two to three ordersof magnitude lower than similarly sized proteins. (Lynch et al., Hum.Gene. Ther.; 4:259-72 (1993). The poor expression of FVIII is due inpart to the presence of cis-acting elements in the FVIII coding sequencethat inhibit FVIII expression, such as transcriptional silencer elements(Hoeben et al., Blood 85:2447-2454 (1995)), matrix attachment-likesequences (MARs) (Fallux et al., Mol. Cell. Biol. 16:4264-4272 (1996)),and transcriptional elongation inhibitory elements (Koeberl et al., Hum.Gene. Ther.; 6:469-479 (1995)).

Advances in our understanding of the biology of FVIII expression has ledto the development of more potent FVIII variants. For instance,biochemical studies demonstrated that the FVIII B-domain was dispensablefor FVIII cofactor activity. Deletion of the B-domain resulted in a17-fold increase in mRNA levels over full-length wild-type FVIII and a30% increase in secreted protein. (Toole et al., Proc Natl Acad Sci USA83:5939-42 (1986)). This led to the development of B domain-deleted(BDD) FVIII protein concentrate, which is now widely used in the clinic.Recent studies, however, indicate that full length and BDD hFVIIImisfold in the ER lumen, resulting in activation of the unfolded proteinresponse (UPR) and apoptosis of murine hepatocytes.

Thus, there exists a need in the art for FVIII sequences that expressefficiently in heterologous systems.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence at least 85% identical to SEQ ID NO:1,wherein the nucleotide sequence encodes a polypeptide with Factor VIIIactivity. In one embodiment, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence at least 90% identical toSEQ ID NO:1. In another embodiment, the invention provides an isolatednucleic acid molecule comprising a nucleotide sequence at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 100%identical to SEQ ID NO:1. In other embodiments, the invention providesan isolated nucleic acid molecule comprising SEQ ID NO:1.

The present invention also provides an isolated nucleic acid moleculecomprising a nucleotide sequence at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or at least 100% identical to SEQ IDNO:2, wherein the nucleotide sequence encodes a polypeptide with FactorVIII activity. In one embodiment, the invention provides an isolatednucleic acid molecule comprising SEQ ID NO:2.

In some embodiments, the isolated nucleic acid molecule of the inventionhas a human codon adaptation index that is increased relative to SEQ IDNO:3. In other embodiments, the isolated nucleic acid molecule of theinvention has a human codon adaptation index that is at least about0.75, at least about 0.76, at least about 0.77, at least about 0.78, atleast about 0.79, or at least about 0.80. In still other embodiments,the isolated nucleic acid molecule of the invention has a human codonadaptation index that is at least about 0.80, at least about 0.81, atleast about 0.82, at least about 0.83, at least about 0.84, at leastabout 0.85, at least about 0.86, at least about 0.87, or at least about0.88.

In certain embodiments, the isolated nucleic acid molecule of theinvention contains a higher percentage of G/C nucleotides compared tothe percentage of G/C nucleotides in SEQ ID NO:3. In other embodiments,the isolated nucleic acid molecule of the invention contains apercentage of G/C nucleotides that is at least about 45%, at least about46%, at least about 47%, at least about 48%, at least about 49%, or atleast about 50%.

In still other embodiments, the isolated nucleic acid molecule of theinvention contains fewer MARS/ARS sequences (SEQ ID NOs: 5 and 6)relative to SEQ ID NO:3. In yet other embodiments, the isolated nucleicacid molecule of the invention contains at most one MARS/ARS sequence.In some embodiments, the isolated nucleic acid molecule of the inventiondoes not contain a MARS/ARS sequence.

In some embodiments, the isolated nucleic acid molecule of the inventiondoes not contain the splice site GGTGAT (SEQ ID NO:7).

In certain embodiments, the isolated nucleic acid molecule of theinvention contains fewer destabilizing sequences (SEQ ID NOs: 8 and 9)relative to SEQ ID NO:3. In other embodiments, the isolated nucleic acidmolecule of the invention contains at most 4 destabilizing sequences. Instill other embodiments, the isolated nucleic acid molecule of theinvention contains at most 2 destabilizing sequences. In yet otherembodiments, the isolated nucleic acid molecule of the invention doesnot contain a destabilizing sequence.

In other embodiments, the isolated nucleic acid molecule of theinvention does not contain a poly-T sequence (SEQ ID NO:10). In yetother embodiments, the isolated nucleic acid molecule of the inventiondoes not contain a poly-A sequence (SEQ ID NO:11).

In one embodiment, the isolated nucleic acid molecule of the inventionfurther comprises a heterologous nucleotide sequence. For example, theheterologous nucleotide sequence can encode a heterologous amino acidsequence that is a half-life extender. In some embodiments, theheterologous amino acid sequence is an immunoglobulin constant region ora portion thereof, transferrin, albumin, albumin-binding polypeptide, anXTEN sequence, Fc, the C-terminal peptide (CTP) of the β subunit ofhuman chorionic gonadotropin, or a PAS sequence. In other embodiments,the heterologous amino acid sequence is an Fc region or an FcRn bindingpartner. In still other embodiments, the heterologous amino acidsequence is linked to the N-terminus or the C-terminus of the amino acidsequence encoded by the nucleotide sequence or inserted between twoamino acids in the amino acid sequence encoded by the nucleotidesequence.

In a particular embodiment, the isolated nucleic acid molecule of theinvention encodes a monomer-dimer hybrid molecule comprising FactorVIII.

In another embodiment, the isolated nucleic acid molecule of theinvention is operatively linked to at least one transcription controlsequence.

The present invention also provides a vector comprising the nucleic acidmolecule of the invention.

The present invention also provides a host cell comprising the nucleicacid molecule of the invention. In some embodiments, the host cell isselected from the group consisting of: a CHO cell, a HEK293 cell, aBHK21 cell, a PER.C6 cell, a NS0 cell, and a CAP cell.

The present invention also provides a polypeptide encoded by the nucleicacid molecule of the invention or the vector of the invention orproduced by the host cell of the invention.

The present invention also provides a method of producing a polypeptidewith Factor VIII activity, comprising: culturing the host cell of theinvention under conditions whereby a polypeptide with Factor VIIIactivity is produced; and, recovering the polypeptide with Factor VIIIactivity. In other embodiments of the method of producing a polypeptidewith Factor VIII activity, the expression of the polypeptide with FactorVIII activity is increased relative to a host cell cultured under thesame conditions comprising a reference nucleotide sequence comprisingSEQ ID NO: 3. In other embodiments of the method, the host cell is a CHOcell. In other embodiments of the method, the host cell is a HEK293cell.

The present invention also provides a method of increasing expression ofa polypeptide with Factor VIII activity in a subject comprisingadministering the isolated nucleic acid molecule of the invention or thevector of the invention to a subject in need thereof, wherein theexpression of the polypeptide with Factor VIII activity is increasedrelative to a reference nucleic acid molecule comprising SEQ ID NO: 3 orthe vector comprising the reference nucleic acid molecule.

The present invention also provides a method of increasing expression ofa polypeptide with Factor VIII activity comprising culturing the hostcell of the invention under conditions whereby a polypeptide with FactorVIII activity is expressed by the nucleic acid molecule, wherein theexpression of the polypeptide with Factor VIII activity is increasedrelative to a host cell cultured under the same conditions comprising areference nucleic acid sequence comprising SEQ ID NO: 3.

The present invention also provides a method of improving yield of apolypeptide with Factor VIII activity comprising culturing the host cellof the invention under conditions whereby a polypeptide with Factor VIIIactivity is produced by the nucleic acid molecule, wherein the yield ofthe polypeptide with Factor VIII activity is increased relative to ahost cell cultured under the same conditions comprising a referencenucleic acid sequence comprising SEQ ID NO: 3.

The present invention also provides a method of treating a bleedingdisorder comprising: administering to a subject in need thereof anucleic acid molecule of the invention, a vector of the invention, or apolypeptide of the invention. In some embodiments of the method oftreating a bleeding disorder, the bleeding disorder is characterized bya deficiency in Factor VIII. In some embodiments, the bleeding disorderis hemophilia. In some embodiments, the bleeding disorder is hemophiliaA.

In some embodiments of the method of treating a bleeding disorder,plasma Factor VIII activity at 24 hours post administration is increasedrelative to a subject administered a reference nucleic acid moleculecomprising SEQ ID NO: 3, a vector comprising the reference nucleic acidmolecule, or a polypeptide encoded by the reference nucleic acidmolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the location of various sites inthe BDD Factor VIII coding sequence. These sites were removed during thecodon optimization process.

FIG. 2 is the nucleotide sequence of BDD Factor VIII (SEQ ID NO:1),codon optimized by a first codon optimization method, described inExample 1.

FIG. 3 is the nucleotide sequence of BDD Factor VIII (SEQ ID NO:2),codon optimized by a second codon optimization method, described inExample 2.

FIGS. 4 A-B show the codon usage bias adjustment in the optimized BDDFVIII sequence (SEQ ID NO: 1). FIG. 4A shows the relative frequency ofcodons in the BDD FVIII sequence before codon optimization. The humancodon adaptation index (CAI) of the starting BDD FVIII sequence is 0.74.FIG. 4B shows the relative frequency of codons in the optimized BDDFVIII sequence (SEQ ID NO:1). The human CAI of the resulting optimizedsequence is 0.88. The X-axis indicates the relative position of thecodons along the length of the BDD FVIII nucleotide sequence. The Y-axisindicates the relative frequency of the codon at each position withinthe human genome.

FIGS. 5 A-B show the frequency of optimal human codons in the optimizedBDD FVIII sequence (SEQ ID NO:1). FIG. 5A shows the frequency of optimalcodons in the BDD FVIII sequence before codon optimization. FIG. 5Bshows the frequency of optimal codons in the BDD FVIII sequence aftercodon optimization (SEQ ID NO:1). The X-axis indicates codon frequencyin the human genome. The Y-axis indicates the percentage of codons inthe BDD FVIII sequence that fall into each category delineated on theX-axis.

FIGS. 6 A-B shows the G/C content of the optimized BDD FVIII sequence(SEQ ID NO:1). FIG. 6A shows the G/C content of the BDD FVIII sequencebefore codon optimization. The G/C content of the starting BDD FVIIIsequence is 46.16%. FIG. 6B shows the G/C content of the BDD FVIIIsequence after codon optimization (SEQ ID NO:1). The G/C content of theoptimized BDD FVIII sequence is 51.56%. The X-axis indicates therelative position of the codons along the length of the BDD FVIIInucleotide sequence. The Y-axis indicates the percent G/C content.

FIG. 7 is a histogram showing plasma FVIII activity in HemA mice 24hours post hydrodynamic injection with plasmids containing either thestarting BDD FVIII sequence (circles), optimized BDD FVIII sequence (SEQID NO:1) (squares), or optimized BDD FVIII sequence (SEQ ID NO:2)(triangles).

DETAILED DESCRIPTION OF THE INVENTION

Exemplary constructs of the invention are illustrated in theaccompanying Figures and sequence listing. In order to provide a clearunderstanding of the specification and claims, the following definitionsare provided below.

I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity: for example, “a nucleotide sequence” is understood torepresent one or more nucleotide sequences. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10 percent, up or down (higher or lower).

The term “isolated” for the purposes of the present invention designatesa biological material (cell, nucleic acid or protein) that has beenremoved from its original environment (the environment in which it isnaturally present). For example, a polynucleotide present in the naturalstate in a plant or an animal is not isolated, however the samepolynucleotide separated from the adjacent nucleic acids in which it isnaturally present, is considered “isolated.”

“Nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” and“polynucleotide” are used interchangeably and refer to the phosphateester polymeric form of ribonucleosides (adenosine, guanosine, uridineor cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), orany phosphoester analogs thereof, such as phosphorothioates andthioesters, in either single stranded form, or a double-stranded helix.Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. Theterm nucleic acid molecule, and in particular DNA or RNA molecule,refers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear or circularDNA molecules (e.g., restriction fragments), plasmids, supercoiled DNAand chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences can be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the non-transcribed strand of DNA (i.e., thestrand having a sequence homologous to the mRNA). A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation. DNA includes, but is not limited to, cDNA, genomic DNA,plasmid DNA, synthetic DNA, and semi-synthetic DNA. A “nucleic acidcomposition” of the invention comprises one or more nucleic acids asdescribed herein.

As used herein, a “coding region” or “coding sequence” is a portion ofpolynucleotide which consists of codons translatable into amino acids.Although a “stop codon” (TAG, TGA, or TAA) is typically not translatedinto an amino acid, it can be considered to be part of a coding region,but any flanking sequences, for example promoters, ribosome bindingsites, transcriptional terminators, introns, and the like, are not partof a coding region. The boundaries of a coding region are typicallydetermined by a start codon at the 5′ terminus, encoding the aminoterminus of the resultant polypeptide, and a translation stop codon atthe 3′ terminus, encoding the carboxyl terminus of the resultingpolypeptide. Two or more coding regions can be present in a singlepolynucleotide construct, e.g., on a single vector, or in separatepolynucleotide constructs, e.g., on separate (different) vectors. Itfollows, then, that a single vector can contain just a single codingregion, or comprise two or more coding regions.

Certain proteins secreted by mammalian cells are associated with asecretory signal peptide which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated, Those of ordinary skill in the art areaware that signal peptides are generally fused to the N-terminus of thepolypeptide, and are cleaved from the complete or “full-length”polypeptide to produce a secreted or “mature” form of the polypeptide.In certain embodiments, a native signal peptide or a functionalderivative of that sequence that retains the ability to direct thesecretion of the polypeptide that is operably associated with it.Alternatively, a heterologous mammalian signal peptide, e.g., a humantissue plasminogen activator (TPA) or mouse β-glucuronidase signalpeptide, or a functional derivative thereof, can be used.

The term “downstream” refers to a nucleotide sequence that is located 3′to a reference nucleotide sequence. In certain embodiments, downstreamnucleotide sequences relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence. In certain embodiments, upstreamnucleotide sequences relate to sequences that are located on the 5′ sideof a coding region or starting point of transcription. For example, mostpromoters are located upstream of the start site of transcription.

As used herein, the term “regulatory region” refers to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding region, and whichinfluence the transcription, RNA processing, stability, or translationof the associated coding region. Regulatory regions can includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures. If a coding region is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

A polynucleotide which encodes a gene product, e.g., a polypeptide, caninclude a promoter and/or other transcription or translation controlelements operably associated with one or more coding regions. In anoperable association a coding region for a gene product, e.g., apolypeptide, is associated with one or more regulatory regions in such away as to place expression of the gene product under the influence orcontrol of the regulatory region(s). For example, a coding region and apromoter are “operably associated” if induction of promoter functionresults in the transcription of mRNA encoding the gene product encodedby the coding region, and if the nature of the linkage between thepromoter and the coding region does not interfere with the ability ofthe promoter to direct the expression of the gene product or interferewith the ability of the DNA template to be transcribed. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can also be operably associated with a coding region to direct geneproduct expression.

“Transcriptional control sequences” refer to DNA regulatory sequences,such as promoters, enhancers, terminators, and the like, that providefor the expression of a coding sequence in a host cell. A variety oftranscription control regions are known to those skilled in the art.These include, without limitation, transcription control regions whichfunction in vertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (the immediate early promoter,in conjunction with intron-A), simian virus 40 (the early promoter), andretroviruses (such as Rous sarcoma virus). Other transcription controlregions include those derived from vertebrate genes such as actin, heatshock protein, bovine growth hormone and rabbit β-globin, as well asother sequences capable of controlling gene expression in eukaryoticcells. Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as lymphokine-induciblepromoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

The term “expression” as used herein refers to a process by which apolynucleotide produces a gene product, for example, an RNA or apolypeptide. It includes without limitation transcription of thepolynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), smallhairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNAproduct, and the translation of an mRNA into a polypeptide. Expressionproduces a “gene product.” As used herein, a gene product can be eithera nucleic acid, e.g., a messenger RNA produced by transcription of agene, or a polypeptide which is translated from a transcript. Geneproducts described herein further include nucleic acids with posttranscriptional modifications, e.g., polyadenylation or splicing, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, or proteolytic cleavage. The term “yield,” as used herein,refers to the amount of a polypeptide produced by the expression of agene.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector can be a replicon to whichanother nucleic acid segment can be attached so as to bring about thereplication of the attached segment. A “replicon” refers to any geneticelement (e.g., plasmid, phage, cosmid, chromosome, virus) that functionsas an autonomous unit of replication in vivo, i.e., capable ofreplication under its own control. The term “vector” includes both viraland nonviral vehicles for introducing the nucleic acid into a cell invitro, ex vivo or in vivo. A large number of vectors are known and usedin the art including, for example, plasmids, modified eukaryoticviruses, or modified bacterial viruses. Insertion of a polynucleotideinto a suitable vector can be accomplished by ligating the appropriatepolynucleotide fragments into a chosen vector that has complementarycohesive termini.

Vectors can be engineered to encode selectable markers or reporters thatprovide for the selection or identification of cells that haveincorporated the vector. Expression of selectable markers or reportersallows identification and/or selection of host cells that incorporateand express other coding regions contained on the vector. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like. Examples of reporters knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ),-glucuronidase (Gus), and the like. Selectable markers can also beconsidered to be reporters.

The term “selectable marker” refers to an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” refers to a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes can also beconsidered reporter genes.

“Promoter” and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the expression of a codingsequence or functional RNA. In general, a coding sequence is located 3′to a promoter sequence. Promoters can be derived in their entirety froma native gene, or be composed of different elements derived fromdifferent promoters found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters can direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters.” Promoters that cause agene to be expressed in a specific cell type are commonly referred to as“cell-specific promoters” or “tissue-specific promoters.” Promoters thatcause a gene to be expressed at a specific stage of development or celldifferentiation are commonly referred to as “developmentally-specificpromoters” or “cell differentiation-specific promoters.” Promoters thatare induced and cause a gene to be expressed following exposure ortreatment of the cell with an agent, biological molecule, chemical,ligand, light, or the like that induces the promoter are commonlyreferred to as “inducible promoters” or “regulatable promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths can have identical promoter activity.

The promoter sequence is typically bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The terms “restriction endonuclease” and “restriction enzyme” are usedinterchangeably and refer to an enzyme that binds and cuts within aspecific nucleotide sequence within double stranded DNA.

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements can be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

Eukaryotic viral vectors that can be used include, but are not limitedto, adenovirus vectors, retrovirus vectors, adeno-associated virusvectors, poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, orherpesvirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid that replicates sequentially and which comprises an originof replication, such as a plasmid, phage or cosmid, to which anothernucleic acid segment can be attached so as to bring about thereplication of the attached segment. Certain cloning vectors are capableof replication in one cell type, e.g., bacteria and expression inanother, e.g., eukaryotic cells. Cloning vectors typically comprise oneor more sequences that can be used for selection of cells comprising thevector and/or one or more multiple cloning sites for insertion ofnucleic acid sequences of interest.

The term “expression vector” refers to a vehicle designed to enable theexpression of an inserted nucleic acid sequence following insertion intoa host cell. The inserted nucleic acid sequence is placed in operableassociation with regulatory regions as described above.

Vectors are introduced into host cells by methods well known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter.

“Culture,” “to culture” and “culturing,” as used herein, means toincubate cells under in vitro conditions that allow for cell growth ordivision or to maintain cells in a living state. “Cultured cells,” asused herein, means cells that are propagated in vitro.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a natural biological source or producedrecombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

The term “amino acid” includes alanine (Ala or A); arginine (Arg or R);asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C);glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G);histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine(Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline(Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp orW); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional aminoacids are also within the scope of the invention and include norleucine,omithine, norvaline, homoserine, and other amino acid residue analoguessuch as those described in Ellman et al. Meth. Enzym. 202:301-336(1991). To generate such non-naturally occurring amino acid residues,the procedures of Noren et al. Science 244:182 (1989) and Ellman et al.,supra, can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.Introduction of the non-traditional amino acid can also be achievedusing peptide chemistries known in the art. As used herein, the term“polar amino acid” includes amino acids that have net zero charge, buthave non-zero partial charges in different portions of their side chains(e.g. M, F, W, S, Y, N, Q, C). These amino acids can participate inhydrophobic interactions and electrostatic interactions. As used herein,the term “charged amino acid” includes amino acids that can havenon-zero net charge on their side chains (e.g. R, K, H, E, D). Theseamino acids can participate in hydrophobic interactions andelectrostatic interactions.

An “isolated” polypeptide or a fragment, variant, or derivative thereofrefers to a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan simply be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for the purpose of the invention, as are nativeor recombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included in the present invention are fragments or variants ofpolypeptides, and any combination thereof. The term “fragment” or“variant” when referring to polypeptide binding domains or bindingmolecules of the present invention include any polypeptides which retainat least some of the properties (e.g., FcRn binding affinity for an FcRnbinding domain or Fc variant, coagulation activity for an FVIII variant,or FVIII binding activity for the VWF fragment) of the referencepolypeptide. Fragments of polypeptides include proteolytic fragments, aswell as deletion fragments, in addition to specific antibody fragmentsdiscussed elsewhere herein, but do not include the naturally occurringfull-length polypeptide (or mature polypeptide). Variants of polypeptidebinding domains or binding molecules of the present invention includefragments as described above, and also polypeptides with altered aminoacid sequences due to amino acid substitutions, deletions, orinsertions. Variants can be naturally or non-naturally occurring.Non-naturally occurring variants can be produced using art-knownmutagenesis techniques. Variant polypeptides can comprise conservativeor non-conservative amino acid substitutions, deletions or additions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, if an amino acid in apolypeptide is replaced with another amino acid from the same side chainfamily, the substitution is considered to be conservative. In anotherembodiment, a string of amino acids can be conservatively replaced witha structurally similar string that differs in order and/or compositionof side chain family members.

The term “percent identity” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case can be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in: Computational Molecular Biology (Lesk, A. M., ed.)Oxford University Press, New York (1988); Biocomputing: Informatics andGenome Projects (Smith, D. W., ed.) Academic Press, New York (1993);Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin,H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, New York (1991). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity are codified in publicly available computer programs.Sequence alignments and percent identity calculations can be performedusing sequence analysis software such as the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.),the GCG suite of programs (Wisconsin Package Version 9.0, GeneticsComputer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschulet al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228S. Park St. Madison, Wis. 53715 USA). Within the context of thisapplication it will be understood that where sequence analysis softwareis used for analysis, that the results of the analysis will be based onthe “default values” of the program referenced, unless otherwisespecified. As used herein “default values” will mean any set of valuesor parameters which originally load with the software when firstinitialized. For the purposes of determining percent identity between anoptimized BDD FVIII sequence of the invention and a reference sequence,only nucleotides in the reference sequence corresponding to nucleotidesin the optimized BDD FVIII sequence of the invention are used tocalculate percent identity. For example, when comparing a full lengthFVIII nucleotide sequence containing the B domain to an optimized Bdomain deleted (BDD) FVIII nucleotide sequence of the invention, theportion of the alignment including the A1, A2, A3, C1, and C2 domainwill be used to calculate percent identity. The nucleotides in theportion of the full length FVIII sequence encoding the B domain (whichwill result in a large “gap” in the alignment) will not be counted as amismatch.

As used herein, “nucleotides corresponding to nucleotides in theoptimized BDD FVIII sequence of the invention” are identified byalignment of the optimized BDD FVIII sequence of the invention tomaximize the identity to the reference FVIII sequence. The number usedto identify an equivalent amino acid in a reference FVIII sequence isbased on the number used to identify the corresponding amino acid in theoptimized BDD FVIII sequence of the invention.

A “fusion” or “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences which normally exist inseparate proteins can be brought together in the fusion polypeptide, orthe amino acid sequences which normally exist in the same protein can beplaced in a new arrangement in the fusion polypeptide, e.g., fusion of aFactor VIII domain of the invention with an Ig Fc domain. A fusionprotein is created, for example, by chemical synthesis, or by creatingand translating a polynucleotide in which the peptide regions areencoded in the desired relationship. A chimeric protein can furthercomprises a second amino acid sequence associated with the first aminoacid sequence by a covalent, non-peptide bond or a non-covalent bond.

As used herein, the term “half-life” refers to a biological half-life ofa particular polypeptide in vivo. Half-life can be represented by thetime required for half the quantity administered to a subject to becleared from the circulation and/or other tissues in the animal. When aclearance curve of a given polypeptide is constructed as a function oftime, the curve is usually biphasic with a rapid α-phase and longerβ-phase. The α-phase typically represents an equilibration of theadministered Fc polypeptide between the intra- and extra-vascular spaceand is, in part, determined by the size of the polypeptide. The β-phasetypically represents the catabolism of the polypeptide in theintravascular space. In some embodiments, FVIII and chimeric proteinscomprising FVIII are monophasic, and thus do not have an alpha phase,but just the single beta phase. Therefore, in certain embodiments, theterm half-life as used herein refers to the half-life of the polypeptidein the β-phase.

The term “linked” as used herein refers to a first amino acid sequenceor nucleotide sequence covalently or non-covalently joined to a secondamino acid sequence or nucleotide sequence, respectively. The firstamino acid or nucleotide sequence can be directly joined or juxtaposedto the second amino acid or nucleotide sequence or alternatively anintervening sequence can covalently join the first sequence to thesecond sequence. The term “linked” means not only a fusion of a firstamino acid sequence to a second amino acid sequence at the C-terminus orthe N-terminus, but also includes insertion of the whole first aminoacid sequence (or the second amino acid sequence) into any two aminoacids in the second amino acid sequence (or the first amino acidsequence, respectively). In one embodiment, the first amino acidsequence can be linked to a second amino acid sequence by a peptide bondor a linker. The first nucleotide sequence can be linked to a secondnucleotide sequence by a phosphodiester bond or a linker. The linker canbe a peptide or a polypeptide (for polypeptide chains) or a nucleotideor a nucleotide chain (for nucleotide chains) or any chemical moiety(for both polypeptide and polynucleotide chains). The term “linked” isalso indicated by a hyphen (-).

As used herein the term “associated with” refers to a covalent ornon-covalent bond formed between a first amino acid chain and a secondamino acid chain. In one embodiment, the term “associated with” means acovalent, non-peptide bond or a non-covalent bond. This association canbe indicated by a colon, i.e., (:). In another embodiment, it means acovalent bond except a peptide bond. For example, the amino acidcysteine comprises a thiol group that can form a disulfide bond orbridge with a thiol group on a second cysteine residue. In mostnaturally occurring IgG molecules, the CH1 and CL regions are associatedby a disulfide bond and the two heavy chains are associated by twodisulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).Examples of covalent bonds include, but are not limited to, a peptidebond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, api bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond,a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruplebond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation,aromaticity, hapticity, or antibonding. Non-limiting examples ofnon-covalent bond include an ionic bond (e.g., cation-pi bond or saltbond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogencomplex, low-barrier hydrogen bond, or symmetric hydrogen bond), van derWalls force, London dispersion force, a mechanical bond, a halogen bond,aurophilicity, intercalation, stacking, entropic force, or chemicalpolarity.

The term “monomer-dimer hybrid” used herein refers to a chimeric proteincomprising a first polypeptide chain and a second polypeptide chain,which are associated with each other by a disulfide bond, wherein thefirst chain comprises a clotting factor, e.g., Factor VIII, and a firstFc region and the second chain comprises, consists essentially of, orconsists of a second Fc region without the clotting factor. Themonomer-dimer hybrid construct thus is a hybrid comprising a monomeraspect having only one clotting factor and a dimer aspect having two Fcregions.

Hemostasis, as used herein, means the stopping or slowing of bleeding orhemorrhage; or the stopping or slowing of blood flow through a bloodvessel or body part.

Hemostatic disorder, as used herein, means a genetically inherited oracquired condition characterized by a tendency to hemorrhage, eitherspontaneously or as a result of trauma, due to an impaired ability orinability to form a fibrin clot. Examples of such disorders include thehemophilias. The three main forms are hemophilia A (factor VIIIdeficiency), hemophilia B (factor IX deficiency or “Christmas disease”)and hemophilia C (factor XI deficiency, mild bleeding tendency). Otherhemostatic disorders include, e.g., von Willebrand disease, Factor XIdeficiency (PTA deficiency), Factor XII deficiency, deficiencies orstructural abnormalities in fibrinogen, prothrombin, Factor V, FactorVII, Factor X or factor XIII, Bernard-Soulier syndrome, which is adefect or deficiency in GPIb. GPIb, the receptor for vWF, can bedefective and lead to lack of primary clot formation (primaryhemostasis) and increased bleeding tendency), and thrombasthenia ofGlanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acuteand chronic forms), there is insufficient production of coagulationfactors by the liver; this can increase bleeding risk.

The isolated nucleic acid molecules or polypeptides of the invention canbe used prophylactically. As used herein the term “prophylactictreatment” refers to the administration of a molecule prior to ableeding episode. In one embodiment, the subject in need of a generalhemostatic agent is undergoing, or is about to undergo, surgery. Thechimeric protein of the invention can be administered prior to or aftersurgery as a prophylactic. The chimeric protein of the invention can beadministered during or after surgery to control an acute bleedingepisode. The surgery can include, but is not limited to, livertransplantation, liver resection, dental procedures, or stem celltransplantation.

The isolated nucleic acid molecules and polypeptides of the inventionare also used for on-demand treatment. The term “on-demand treatment”refers to the administration of an isolated nucleic acid molecule orpolypeptide in response to symptoms of a bleeding episode or before anactivity that can cause bleeding. In one aspect, the on-demand treatmentcan be given to a subject when bleeding starts, such as after an injury,or when bleeding is expected, such as before surgery. In another aspect,the on-demand treatment can be given prior to activities that increasethe risk of bleeding, such as contact sports.

As used herein the term “acute bleeding” refers to a bleeding episoderegardless of the underlying cause. For example, a subject can havetrauma, uremia, a hereditary bleeding disorder (e.g., factor VIIdeficiency) a platelet disorder, or resistance owing to the developmentof antibodies to clotting factors.

Treat, treatment, treating, as used herein refers to, e.g., thereduction in severity of a disease or condition; the reduction in theduration of a disease course; the amelioration of one or more symptomsassociated with a disease or condition; the provision of beneficialeffects to a subject with a disease or condition, without necessarilycuring the disease or condition, or the prophylaxis of one or moresymptoms associated with a disease or condition. In one embodiment, theterm “treating” or “treatment” means maintaining a FVIII trough level atleast about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7IU/dL, 8 IU/dL, 9 IU/dL, 10 IU/dL, 11 IU/dL, 12 IU/dL, 3 IU/dL, 14IU/dL, 15 IU/dL, 16 IU/dL, 17 IU/dL, 18 IU/dL, 19 IU/dL, or 20 IU/dL ina subject by administering an isolated nucleic acid molecule orpolypeptide of the invention. In another embodiment, treating ortreatment means maintaining a FVIII trough level between about 1 andabout 20 IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL,about 4 and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 andabout 20 IU/dL, about 7 and about 20 IU/dL, about 8 and about 20 IU/dL,about 9 and about 20 IU/dL, or about 10 and about 20 IU/dL. Treatment ortreating of a disease or condition can also include maintaining FVIIIactivity in a subject at a level comparable to at least about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, or 20% of the FVIII activity in a non-hemophiliac subject. Theminimum trough level required for treatment can be measured by one ormore known methods and can be adjusted (increased or decreased) for eachperson.

“Administering,” as used herein, means to give a pharmaceuticallyacceptable Factor VIII polypeptide of the invention to a subject via apharmaceutically acceptable route. Routes of administration can beintravenous, e.g., intravenous injection and intravenous infusion.Additional routes of administration include, e.g., subcutaneous,intramuscular, oral, nasal, and pulmonary administration. Chimericpolypeptides and hybrid proteins can be administered as part of apharmaceutical composition comprising at least one excipient.

As used herein, the phrase “subject in need thereof” includes subjects,such as mammalian subjects, that would benefit from administration of anucleic acid molecule or a polypeptide of the invention, e.g., toimprove hemostasis. In one embodiment, the subjects include, but are notlimited to, individuals with hemophilia. In another embodiment, thesubjects include, but are not limited to, the individuals who havedeveloped a FVIII inhibitor and thus are in need of a bypass therapy.The subject can be an adult or a minor (e.g., under 12 years old).

As used herein, the term “clotting factor,” refers to molecules, oranalogs thereof, naturally occurring or recombinantly produced whichprevent or decrease the duration of a bleeding episode in a subject. Inother words, it means molecules having pro-clotting activity, i.e., areresponsible for the conversion of fibrinogen into a mesh of insolublefibrin causing the blood to coagulate or clot. An “activatable clottingfactor” is a clotting factor in an inactive form (e.g., in its zymogenform) that is capable of being converted to an active form.

Clotting activity, as used herein, means the ability to participate in acascade of biochemical reactions that culminates in the formation of afibrin clot and/or reduces the severity, duration or frequency ofhemorrhage or bleeding episode.

As used herein the terms “heterologous” or “exogenous” refer to suchmolecules that are not normally found in a given context, e.g., in acell or in a polypeptide. For example, an exogenous or heterologousmolecule can be introduced into a cell and are only present aftermanipulation of the cell, e.g., by transfection or other forms ofgenetic engineering or a heterologous amino acid sequence can be presentin a protein in which it is not naturally found.

As used herein, the term “heterologous nucleotide sequence” refers to anucleotide sequence that does not naturally occur with a givenpolynucleotide sequence. In one embodiment, the heterologous nucleotidesequence encodes a polypeptide capable of extending the half-life ofFVIII. In another embodiment, the heterologous nucleotide sequenceencodes a polypeptide that increases the hydrodynamic radius of FVIII.In other embodiments, the heterologous nucleotide sequence encodes apolypeptide that improves one or more pharmacokinetic properties ofFVIII without significantly affecting its biological activity orfunction (e.g., its procoagulant activity). In some embodiments, FVIIIis linked or connected to the polypeptide encoded by the heterologousnucleotide sequence by a linker. Non-limiting examples of polypeptidemoieties encoded by heterologous nucleotide sequences include animmunoglobulin constant region or a portion thereof, albumin or afragment thereof, an albumin-binding moiety, a transferrin, the PASpolypeptides of U.S. Pat Application No. 20100292130, a HAP sequence,transferrin or a fragment thereof, the C-terminal peptide (CTP) of the βsubunit of human chorionic gonadotropin, albumin-binding small molecule,an XTEN sequence, FcRn binding moieties (e.g., complete Fc regions orportions thereof which bind to FcRn), single chain Fc regions (ScFcregions, e.g., as described in US 2008/0260738, WO 2008/012543, or WO2008/1439545), polyglycine linkers, polyserine linkers, peptides andshort polypeptides of 6-40 amino acids of two types of amino acidsselected from glycine (G), alanine (A), serine (S), threonine (T),glutamate (E) and proline (P) with varying degrees of secondarystructure from less than 50% to greater than 50%, amongst others, or twoor more combinations thereof. In some embodiments, the polypeptideencoded by the heterologous nucleotide sequence is linked to anon-polypeptide moiety. Non-limiting examples of the non-polypeptidemoieties include polyethylene glycol (PEG), albumin-binding smallmolecules, polysialic acid, hydroxyethyl starch (HES), a derivativethereof, or any combinations thereof.

As used herein, the term “Fc region” is defined as the portion of apolypeptide which corresponds to the Fc region of native Ig, i.e., asformed by the dimeric association of the respective Fc domains of itstwo heavy chains. A native Fc region forms a homodimer with another Fcregion. In contrast, the term “genetically-fused Fc region” or“single-chain Fc region” (scFc region), as used herein, refers to asynthetic dimeric Fc region comprised of Fc domains genetically linkedwithin a single polypeptide chain (i.e., encoded in a single contiguousgenetic sequence).

In one embodiment, the “Fc region” refers to the portion of a single Igheavy chain beginning in the hinge region just upstream of the papaincleavage site (i.e. residue 216 in IgG, taking the first residue ofheavy chain constant region to be 114) and ending at the C-terminus ofthe antibody. Accordingly, a complete Fc domain comprises at least ahinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an Ig constant region, depending on the Ig isotype caninclude the CH2, CH3, and CH4 domains, as well as the hinge region.Chimeric proteins comprising an Fc region of an Ig bestow severaldesirable properties on a chimeric protein including increasedstability, increased serum half-life (see Capon et al., 1989, Nature337:525) as well as binding to Fc receptors such as the neonatal Fcreceptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO03/077834; US2003-0235536A1), which are incorporated herein by referencein their entireties.

A “reference nucleotide sequence,” when used herein as a comparison to anucleotide sequence of the invention, is a polynucleotide sequenceessentially identical to the nucleotide sequence of the invention exceptthat the portions corresponding to FVIII sequence are not optimized. Forexample, the reference nucleotide sequence for a nucleic acid moleculeconsisting of the codon optimized BDD FVIII of SEQ ID NO:1 and aheterologous nucleotide sequence that encodes a single chain Fc regionlinked to SEQ ID NO:1 at its 3′ end is a nucleic acid moleculeconsisting of the original (or “parent”) BDD FVIII of SEQ ID NO:3 andthe identical heterologous nucleotide sequence that encodes a singlechain Fc region linked to SEQ ID NO:3 at its 3′ end.

A “codon adaptation index,” as used herein, refers to a measure of codonusage bias. A codon adaptation index (CAI) measures the deviation of agiven protein coding gene sequence with respect to a reference set ofgenes (Sharp PM and Li WH, Nucleic Acids Res. 15(3):1281-95 (1987)). CAIis calculated by determining the geometric mean of the weight associatedto each codon over the length of the gene sequence (measured in codons):

$\begin{matrix}{{{CAI} = {\exp ( {{1/L}{\sum\limits_{l = 1}^{L}{\ln ( {\omega_{i}(l)} )}}} )}},} & (I)\end{matrix}$

For each amino acid, the weight of each of its codons, in CAI, iscomputed as the ratio between the observed frequency of the codon (fi)and the frequency of the synonymous codon (fj) for that amino acid:

$\begin{matrix}{{{Formula}\mspace{14mu} 2}\mspace{610mu}} & \; \\{\omega_{i} = {{\frac{f_{i}}{\max ( f_{j} )}{ij}} \in \lbrack {{synonymous}\mspace{14mu} {codons}\mspace{14mu} {for}\mspace{14mu} {amino}\mspace{14mu} {acid}} \rbrack}} & ({II})\end{matrix}$

As used herein, the term “optimized,” with regard to nucleotidesequences, refers to a polynucleotide sequence that encodes apolypeptide, wherein the polynucleotide sequence has been mutated toenhance a property of that polynucleotide sequence. In some embodiments,the optimization is done to increase transcription levels, increasetranslation levels, increase steady-state mRNA levels, increase ordecrease the binding of regulatory proteins such as generaltranscription factors, increase or decrease splicing, or increase theyield of the polypeptide produced by the polynucleotide sequence.Examples of changes that can be made to a polynucleotide sequence tooptimize it include codon optimization, G/C content optimization,removal of repeat sequences, removal of AT rich elements, removal ofcryptic splice sites, removal of cis-acting elements that represstranscription or translation, adding or removing poly-T or poly-Asequences, adding sequences around the transcription start site thatenhance transcription, such as Kozak consensus sequences, removal ofsequences that could form stem loop structures, removal of destabilizingsequences, and two or more combinations thereof.

The present invention is directed to optimized Factor VIII sequences,vectors and host cells comprising optimized Factor VIII sequences,polypeptides encoded by optimized Factor VIII sequences, and methods ofproducing such polypeptides. The present invention is also directed tomethods of treating bleeding disorders such as hemophilia comprisingadministering to the subject an optimized Factor VIII nucleic acidsequence or the polypeptide encoded thereby. The present invention meetsan important need in the art by providing optimized Factor VIIIsequences that demonstrate increased expression in host cells, improvedyield of Factor VIII protein in methods to produce recombinant FactorVIII, and potentially result in greater therapeutic efficacy when usedin gene therapy methods.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with Factor VIII (FVIII) activity, wherein the nucleotidesequence is at least 85% identical to SEQ ID NO:1. In other embodiments,the nucleotide sequence is at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to SEQ ID NO:1 and encodes a polypeptide with FVIIIactivity. In still other embodiments, the nucleotide sequence comprisesSEQ ID NO:1.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95% identical to SEQ ID NO:2. In other embodiments, the nucleotidesequence is at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO:2 and encodes a polypeptide with FVIII activity.In still other embodiments, the nucleotide sequence comprises SEQ IDNO:2.

SEQ ID NOs: 1 and 2 are optimized versions of SEQ ID NO:3, the startingor “parental” F VIII nucleotide sequence. SEQ ID NO:3 encodes a Bdomain-deleted human FVIII. While SEQ ID NOs: 1 and 2 are derived from aspecific B domain-deleted form of FVIII (SEQ ID NO:3), it is to beunderstood that the present invention is also directed to optimizedversions of nucleic acids encoding other versions of FVIII. For example,other version of FVIII can include full length FVIII, other B-domaindeletions of FVIII (described below), or other fragments of FVIII thatretain FVIII activity.

“A polypeptide with FVIII activity” as used herein means a functionalFVIII polypeptide in its normal role in coagulation, unless otherwisespecified. The term a polypeptide with FVIII activity includes afunctional fragment, variant, analog, or derivative thereof that retainsthe function of full-length wild-type Factor VIII in the coagulationpathway. “A polypeptide with FVIII activity” is used interchangeablywith FVIII protein, FVIII polypeptide, or FVIII. Examples of FVIIIfunctions include, but are not limited to, an ability to activatecoagulation, an ability to act as a cofactor for factor IX, or anability to form a tenase complex with factor IX in the presence of Ca²⁺and phospholipids, which then converts Factor X to the activated formXa. In one embodiment, a polypeptide having FVIII activity comprises twopolypeptide chains, the first chain having the FVIII heavy chain and thesecond chain having the FVIII light chain. In another embodiment, thepolypeptide having FVIII activity is single chain FVIII. Single chainFVIII can contain one or more mutation or substitutions at amino acidresidue 1645 and/or 1648 corresponding to mature FVIII sequence. SeeInternational Application No. PCT/US2012/045784, incorporated herein byreference in its entirety. The FVIII protein can be the human, porcine,canine, rat, or murine FVIII protein. In addition, comparisons betweenFVIII from humans and other species have identified conserved residuesthat are likely to be required for function (Cameron et al., Thromb.Haemost. 79:317-22 (1998); U.S. Pat. No. 6,251,632).

The “B domain” of FVIII, as used herein, is the same as the B domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage by thrombin, e.g., residuesSer741-Arg1648 of full length human FVIII. The other human FVIII domainsare defined by the following amino acid residues: A1, residuesAla1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Ile2032;C1, residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2sequence includes residues Ser1690-Tyr2332. The remaining sequence,residues Glu1649-Arg1689, is usually referred to as the FVIII lightchain activation peptide. The locations of the boundaries for all of thedomains, including the B domains, for porcine, mouse and canine FVIIIare also known in the art. An example of a BDD FVIII is REFACTO®recombinant BDD FVIII (Wyeth Pharmaceuticals, Inc.).

A “B domain deleted FVIII” can have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In someembodiments, a B domain deleted FVIII sequence of the present inventioncomprises any one of the deletions disclosed at col. 4, line 4 to col.5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S.Pat. No. 6,346,513). In some embodiments, a B domain deleted FVIII ofthe present invention has a deletion disclosed at col. 2, lines 26-51and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. Nos.6,060,447, 5,595,886, and 6,228,620). In some embodiments, a B domaindeleted FVIII has a deletion described in col. 1, lines 25 to col. 2,line 40 of U.S. Pat. No. 5,972,885; col. 6, lines 1-22 and example 1 ofU.S. Pat. No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No. 5,543,502;col. 4, line 22 to col. 5, line 36 of U.S. Pat. No. 5,171,844; col. 2,lines 55-68, FIG. 2, and example 1 of U.S. Pat. No. 5,112,950; col. 2,line 2 to col. 19, line 21 and table 2 of U.S. Pat. No. 4,868,112; col.2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col.7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39of U.S. Pat. No. 7,041,635; or col. 4, lines 25-53, of U.S. Pat. No.6,458,563. In some embodiments, a B domain deleted FVIII has a deletionof most of the B domain, but still contains amino-terminal sequences ofthe B domain that are essential for in vivo proteolytic processing ofthe primary translation product into two polypeptide chain, as disclosedin WO 91/09122, which is incorporated herein by reference in itsentirety. In some embodiments, a B domain deleted FVIII is constructedwith a deletion of amino acids 747-1638, i.e., virtually a completedeletion of the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13):7318-7323 (1990), incorporated herein by reference in its entirety. A Bdomain deleted FVIII can also contain a deletion of amino acids 771-1666or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4):301-6 (1988), incorporated herein by reference in its entirety.Additional B domain deletions that are part of the invention include,e.g.: deletion of amino acids 982 through 1562 or 760 through 1639(Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741through 1646 (Kaufman (PCT published application No. WO 87/04187)),747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 through 1648(Pasek (PCT application No. 88/00831)), 816 through 1598 or 741 through1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), eachof which is incorporated herein by reference in its entirety. Each ofthe foregoing deletions can be made in any FVIII sequence.

A number of functional FVIII molecules, including B-domain deletions,are disclosed in the following patents U.S. Pat. Nos. 6,316,226 and6,346,513, both assigned to Baxter; U.S. Pat. No. 7,041,635 assigned toIn2Gen; U.S. Pat. Nos. 5,789,203, 6,060,447, 5,595,886, and 6,228,620assigned to Chiron; U.S. Pat. Nos. 5,972,885 and 6,048,720 assigned toBiovitrum, U.S. Pat. Nos. 5,543,502 and 5,610,278 assigned to NovoNordisk; U.S. Pat. No. 5,171,844 assigned to Immuno Ag; U.S. Pat. No.5,112,950 assigned to Transgene S. A.; U.S. Pat. No. 4,868,112 assignedto Genetics Institute, each of which is incorporated herein by referencein its entirety.

Codon Optimization

In one embodiment, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleic acid sequence hasbeen codon optimized. In another embodiment, the starting nucleic acidsequence that encodes a polypeptide with FVIII activity and that issubject to codon optimization is SEQ ID NO:3. In some embodiments, thesequence that encodes a polypeptide with FVIII activity is codonoptimized for human expression. In other embodiments, the sequence thatencodes a polypeptide with FVIII activity is codon optimized for murineexpression. SEQ ID NOs: 1 and 2 are codon optimized versions of SEQ IDNO:3, optimized for human expression.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

Deviations in the nucleotide sequence that comprises the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). The “genetic code” which shows which codons encodewhich amino acids is reproduced herein as Table 1. As a result, manyamino acids are designated by more than one codon. For example, theamino acids alanine and proline are coded for by four triplets, serineand arginine by six, whereas tryptophan and methionine are coded by justone triplet. This degeneracy allows for DNA base composition to varyover a wide range without altering the amino acid sequence of theproteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Stop TGA Stop TTG Leu (L) TCG Ser (S) TAG Stop TGG Trp(W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCCPro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGAArg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I)ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N)AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met ACGThr (T) AAG Lys (K) AGG Arg (R) (M) G GTT Val (V) GCT Ala (A) GAT Asp(D) GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val(V) GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu(E) GGG Gly (G)

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing peptide chain. Codonpreference, or codon bias, differences in codon usage between organisms,is afforded by degeneracy of the genetic code, and is well documentedamong many organisms. Codon bias often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, inter alia, the properties of the codons being translatedand the availability of particular transfer RNA (tRNA) molecules. Thepredominance of selected tRNAs in a cell is generally a reflection ofthe codons used most frequently in peptide synthesis. Accordingly, genescan be tailored for optimal gene expression in a given organism based oncodon optimization.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, the relative frequencies of codonusage have been calculated. Codon usage tables are available, forexample, at the “Codon Usage Database” available atwww.kazusa.or.jp/codon/(visited Jun. 18, 2012). See Nakamura, Y., et al.Nucl. Acids Res. 28:292 (2000).

Randomly assigning codons at an optimized frequency to encode a givenpolypeptide sequence can be done manually by calculating codonfrequencies for each amino acid, and then assigning the codons to thepolypeptide sequence randomly. Additionally, various algorithms andcomputer software programs can be used to calculate an optimal sequence.

In one embodiment, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85% identical to SEQ ID NO:1, and wherein the human codonadaptation index is increased relative to SEQ ID NO:3. For example, thenucleotide sequence that encodes a polypeptide with FVIII activity andthat is at least 85% identical to SEQ ID NO:1 can have a human codonadaptation index that is at least about 0.75, at least about 0.76, atleast about 0.77, at least about 0.78, at least about 0.79, at leastabout 0.80, at least about 0.81, at least about 0.82, at least about0.83, at least about 0.84, at least about 0.85, at least about 0.86, atleast about 0.87, at least about 0.88, at least about 0.89, or at leastabout 0.90.

In another embodiment, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with Factor VIII (FVIII) activity, wherein the nucleotidesequence is at least 95% identical to SEQ ID NO:2, and wherein the humancodon adaptation index is increased relative to SEQ ID NO:3. Forexample, the nucleotide sequence that encodes a polypeptide with FVIIIactivity and that is at least 85% identical to SEQ ID NO:2 can have ahuman codon adaptation index that is at least about 0.75, at least about0.76, at least about 0.77, at least about 0.78, at least about 0.79, atleast about 0.80, at least about 0.81, at least about 0.82, at leastabout 0.83, at least about 0.84, at least about 0.85, at least about0.86, at least about 0.87, at least about 0.88, at least about 0.89, orat least about 0.90.

In other embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with Factor VIII activity, wherein the nucleotide sequenceis at least 85% identical to SEQ ID NO:1 and has one or more of thefollowing characteristics: (1) the nucleotide sequence contains a higherpercentage of G/C nucleotides compared to SEQ ID NO:3, (2) thenucleotide sequence contains fewer MARS/ARS sequences compared to SEQ IDNO:3, (3) the nucleotide sequence does not contain the splice siteGGTGAT, (4) the nucleotide sequence contains fewer destabilizingelements, (5) the nucleotide sequence does not contain a poly-Tsequence, (6) the nucleotide sequence does not contain a poly-Asequence, (7) the nucleotide sequence has a codon adaptation index thatis increased relative to SEQ ID NO:3, or a combination of two or moresuch characteristics. In a particular embodiment, the nucleotidesequence contains all of the characteristics (1) to (6).

In other embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with Factor VIII activity, wherein the nucleotide sequenceis at least 95% identical to SEQ ID NO:2 and has one or more of thefollowing characteristics: (1) the nucleotide sequence contains a higherpercentage of G/C nucleotides compared to SEQ ID NO:3, (2) thenucleotide sequence contains fewer MARS/ARS sequences, (3) thenucleotide sequence does not contain the splice site GGTGAT, (4) thenucleotide sequence contains fewer destabilizing elements, (5) thenucleotide sequence does not contain a poly-T sequence, (6) thenucleotide sequence does not contain a poly-A sequence, (7) thenucleotide sequence has a codon adaptation index that is increasedrelative to SEQ ID NO:3, or a combination of two or more suchcharacteristics. In a particular embodiment, the nucleotide sequencecontains all of the characteristics (1) to (6).

G/C Content Optimization

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85% identical to SEQ ID NO:1, and wherein the nucleotide sequencecontains a higher percentage of G/C nucleotides compared to thepercentage of G/C nucleotides in SEQ ID NO:3. In other embodiments, thenucleotide sequence that encodes a polypeptide with FVIII activity andthat is at least 85% identical to SEQ ID NO:1 has a G/C content that isat least about 45%, at least about 46%, at least about 47%, at leastabout 48%, at least about 49%, at least about 50%, at least about 51%,at least about 52%, at least about 53%, at least about 54%, or at leastabout 55%.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95% identical to SEQ ID NO:2, and wherein the nucleotide sequencecontains a higher percentage of G/C nucleotides compared to thepercentage of G/C nucleotides in SEQ ID NO:3. In other embodiments, thenucleotide sequence that encodes a polypeptide with FVIII activity andthat is at least 95% identical to SEQ ID NO:2 has a G/C content that isat least about 45%, at least about 46%, at least about 47%, at leastabout 48%, at least about 49%, at least about 50%, at least about 51%,at least about 52%, at least about 53%, at least about 54%, or at leastabout 55%.

“G/C content” (or guanine-cytosine content), or “percentage of G/Cnucleotides,” refers to the percentage of nitrogenous bases in a DNAmolecule that are either guanine or cytosine. G/C content can becalculated using the following formula:

$\begin{matrix}{\frac{G + C}{A + T + G + C} \times 100} & ({III})\end{matrix}$

Human genes are highly heterogeneous in their G/C content, with somegenes having a G/C content as low as 20%, and other genes having a G/Ccontent as high as 95%. In general, G/C rich genes are more highlyexpressed. In fact, it has been demonstrated that increasing the G/Ccontent of a gene can lead to increased expression of the gene, duemostly to an increase in transcription and higher steady state mRNAlevels. See Kudla et al., PLoS Biol., 4(6): e180 (2006).

Matrix Attachment Region-Like Sequences

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85% identical to SEQ ID NO:1, and wherein the nucleotide sequencecontains fewer MARS/ARS sequences relative to SEQ ID NO:3. In otherembodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity and that is at least 85% identical to SEQ ID NO:1contains at most 6, at most 5, at most 4, at most 3, or at most 2MARS/ARS sequences. In other embodiments, the nucleotide sequence thatencodes a polypeptide with FVIII activity and that is at least 85%identical to SEQ ID NO:1 contains at most 1 MARS/ARS sequence. In yetother embodiments, the nucleotide sequence that encodes a polypeptidewith FVIII activity and that is at least 85% identical to SEQ ID NO: 1does not contain a MARS/ARS sequence.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95% identical to SEQ ID NO:2, and wherein the nucleotide sequencecontains fewer MARS/ARS sequences relative to SEQ ID NO:3. In otherembodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity and that is at least 95% identical to SEQ ID NO:2contains at most 6, at most 5, at most 4, at most 3, or at most 2MARS/ARS sequences. In other embodiments, the nucleotide sequence thatencodes a polypeptide with FVIII activity and that is at least 95%identical to SEQ ID NO:2 contains at most 1 MARS/ARS sequence. In yetother embodiments, the nucleotide sequence that encodes a polypeptidewith FVIII activity and that is at least 95% identical to SEQ ID NO:2does not contain a MARS/ARS sequence.

AT-rich elements in the human FVIII nucleotide sequence that sharesequence similarity with Saccharomyces cerevisiae autonomouslyreplicating sequences (ARSs) and nuclear-matrix attachment regions(MARs) have been identified. (Fallux et al., Mol. Cell. Biol.16:4264-4272 (1996). One of these elements has been demonstrated to bindnuclear factors in vitro and to repress the expression of achloramphenicol acetyltransferase (CAT) reporter gene. Id. It has beenhypothesized that these sequences can contribute to the transcriptionalrepression of the human FVIII gene. Thus, in one embodiment, all MAR/ARSsequences are abolished in the FVIII gene of the present invention.There are four MAR/ARS ATATTT sequences (SEQ ID NO:5) and three MAR/ARSAAATAT sequences (SEQ ID NO:6) in the parental FVIII sequence (SEQ IDNO:3). All of these sites were mutated to destroy the MAR/ARS sequencesin the optimized FVIII sequences (SEQ ID NO:1 and SEQ ID NO:2). Thelocation of each of these elements, and the sequence of thecorresponding nucleotides in the optimized sequences are shown in Table2, below.

TABLE 2 Summary of Changes to Repressive Elements Starting BDD FVIIIOptimized BDD Optimized Location Sequence FVIII BDD FVIII of (SEQSequence Sequence Element ID NO: 3) (SEQ ID NO: 1) (SEQ ID NO: 2) 639ATTTA GTTCA GTTCA 1338 ATTTA GTTCA GTTCA 1449 ATTTA TTTCA CTTCA 1590TAAAT CAAGT CAAGT 1623 TAAAT TAAGA CAAGA 2410 ATTTA ATCTA ATCTA 2586ATTTA GTTTA GTTCA 2630 TAAAT TGAAC TGAAC 3884 ATTTA ATCTG ACCTG 3887TAAAT TGAAC TGAAC Potential Promoter Binding Sites 641 TTATA TCATT TCATC1275 TATAA TACAA TACAA 1276 TTATA CTACA CTACA 1445 TTATA TCATT TCATC1474 TATAA TACAA TACAA 1588 TATAA TACAA TACAA 2614 TTATA CTGTA CTGTA2661 TATAA TATCA CATTA 3286 TATAA TACAA TACAA 3840 TTATA TTATT CTACAMatrix Attachment-Like Sequences (MARS/ARS) 1287 ATATTT GTATCT GTACCT1447 ATATTT ATTTTC ATCTTC 1577 AAATAT AAATCT AGATCT 1585 AAATAT AAGTACAAGTAC 2231 ATATTT ACATCA ACATCA 3054 AAATAT AAACAT GAACAT 3788 ATATTTACATTT ACATCT AU Rich Sequence Elements (AREs) 2468 ATTTTATT ACTTTATTACTTCATT 3790 ATTTTTAA ATTTTCAA ATCTTCAA Poly A/Poly T Sequences 3273AAAAAAA GAAGAAA GAAGAAA 4195 TTTTTT TTCTTT TTCTTT Splice Sites 2203GGTGAT GGGGAC GGCGAC

Destabilizing Sequences

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85% identical to SEQ ID NO:1, and wherein the nucleotide sequencecontains fewer destabilizing elements relative to SEQ ID NO:3. In otherembodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity and that is at least 85% identical to SEQ ID NO:1contains at most 9, at most 8, at most 7, at most 6, or at most 5destabilizing elements. In other embodiments, the nucleotide sequencethat encodes a polypeptide with FVIII activity and that is at least 85%identical to SEQ ID NO:1 contains at most 4, at most 3, at most 2, or atmost 1 destabilizing elements. In yet other embodiments, the nucleotidesequence that encodes a polypeptide with FVIII activity and that is atleast 85% identical to SEQ ID NO:1 does not contain a destabilizingelement.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95% identical to SEQ ID NO:2, and wherein the nucleotide sequencecontains fewer destabilizing elements relative to SEQ ID NO:3. In otherembodiments, the nucleotide sequence that encodes a polypeptide withFVIII activity and that is at least 95% identical to SEQ ID NO:2contains at most 9, at most 8, at most 7, at most 6, or at most 5destabilizing elements. In other embodiments, the nucleotide sequencethat encodes a polypeptide with FVIII activity and that is at least 95%identical to SEQ ID NO:2 contains at most 4, at most 3, at most 2, or atmost 1 destabilizing elements. In yet other embodiments, the nucleotidesequence that encodes a polypeptide with FVIII activity and that is atleast 95% identical to SEQ ID NO:2 does not contain a destabilizingelement.

There are ten destabilizing elements in the parental FVIII sequence (SEQID NO:3); six ATTTA sequences (SEQ ID NO:8) and four TAAAT sequences(SEQ ID NO:9). In one embodiment, sequences of these sites were mutatedto destroy the destabilizing elements in optimized FVIII SEQ ID NO:1 andSEQ ID NO:2. The location of each of these elements, and the sequence ofthe corresponding nucleotides in the optimized sequences are shown inTable 2.

Potential Promoter Binding Sites

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:1, and whereinthe nucleotide sequence contains fewer potential promoter binding sitesrelative to SEQ ID NO:3. In other embodiments, the nucleotide sequencethat encodes a polypeptide with FVIII activity and that is at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to SEQ ID NO:1 contains at most 9, at most8, at most 7, at most 6, or at most 5 potential promoter binding sites.In other embodiments, the nucleotide sequence that encodes a polypeptidewith FVIII activity and that is at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO:1 contains at most 4, at most 3, at most 2, or atmost 1 potential promoter binding sites. In yet other embodiments, thenucleotide sequence that encodes a polypeptide with FVIII activity andthat is at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1 doesnot contain a potential promoter binding site.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO:2, and wherein the nucleotide sequencecontains fewer potential promoter binding sites relative to SEQ ID NO:3.In other embodiments, the nucleotide sequence that encodes a polypeptidewith FVIII activity and that is at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2contains at most 9, at most 8, at most 7, at most 6, or at most 5potential promoter binding sites. In other embodiments, the nucleotidesequence that encodes a polypeptide with FVIII activity and that is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO:2 contains at most 4, at most 3, at most 2,or at most 1 potential promoter binding sites. In yet other embodiments,the nucleotide sequence that encodes a polypeptide with FVIII activityand that is at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to SEQ ID NO:2 does not contain a potentialpromoter binding site.

TATA boxes are regulatory sequences often found in the promoter regionsof eukaryotes. They serve as the binding site of TATA binding protein(TBP), a general transcription factor. TATA boxes usually comprise thesequence TATAAA (SEQ ID NO:12) or a close variant. TATA boxes within acoding sequence, however, can inhibit the translation of full-lengthprotein. There are ten potential promoter binding sequences in the wildtype BDD FVIII sequence (SEQ ID NO:3); five TATAA sequences (SEQ ID NO:12) and five TTATA sequences (SEQ ID NO: 13). In one embodiment, allpromoter binding sites are abolished in the FVIII genes of the presentinvention. The location of each potential promoter binding site and thesequence of the corresponding nucleotides in the optimized sequences areshown in Table 2.

Other Cis Acting Negative Regulatory Elements

In addition to the MAR/ARS sequences, destabilizing elements, andpotential promoter sites described above, several additional potentiallyinhibitory sequences can be identified in the wild type BDD FVIIIsequence (SEQ ID NO:3). Two AU rich sequence elements (AREs) can beidentified (SEQ ID NOs: 14 and 15), along with a poly-A site (SEQ IDNO:11), a poly-T site (SEQ ID NO:10), and a splice site (SEQ ID NO:7) inthe wild type BDD FVIII sequence. One or more of these elements can beremoved from the optimized FVIII sequences. The location of each ofthese sites and the sequence of the corresponding nucleotides in theoptimized sequences are shown in Table 2.

In certain embodiments, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:1, wherein thenucleotide sequence does not contain one or more cis-acting negativeregulatory elements, for example, a splice site, a poly-T sequence, apoly-A sequence, an ARE sequence, or any combinations thereof.

In certain embodiments, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO:2, wherein the nucleotide sequence does notcontain one or more cis-acting negative regulatory elements, forexample, a splice site, a poly-T sequence, a poly-A sequence, an AREsequence, or any combinations thereof.

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and whereinthe nucleotide sequence does not contain the splice site GGTGAT (SEQ IDNO:7). In some embodiments, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:1, and whereinthe nucleotide sequence does not contain a poly-T sequence (SEQ IDNO:10). In some embodiments, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and whereinthe nucleotide sequence does not contain a poly-A sequence (SEQ IDNO:11). In some embodiments, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and whereinthe nucleotide sequence does not contain an ARE element (SEQ ID NO:14 orSEQ ID NO: 15).

In some embodiments, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO:2, and wherein the nucleotide sequence doesnot contain the splice site GGTGAT (SEQ ID NO:7). In some embodiments,the present invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence that encodes a polypeptide with FVIIIactivity, wherein the nucleotide sequence is at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identical to SEQ IDNO:2, and wherein the nucleotide sequence does not contain a poly-Tsequence (SEQ ID NO: 10). In some embodiments, the present inventionprovides an isolated nucleic acid molecule comprising a nucleotidesequence that encodes a polypeptide with FVIII activity, wherein thenucleotide sequence is at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:2, and whereinthe nucleotide sequence does not contain a poly-A sequence (SEQ ID NO:11). In some embodiments, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes apolypeptide with FVIII activity, wherein the nucleotide sequence is atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO:2, and wherein the nucleotide sequence doesnot contain an ARE element (SEQ ID NO:14 or SEQ ID NO:15).

In other embodiments, an optimized FVIII sequence of the invention doesnot comprise one or more of antiviral motifs, stem-loop structures, andrepeat sequences.

In still other embodiments, the nucleotides surrounding thetranscription start site are changed to a kozak consensus sequence(GCCGCCACCATGC, wherein the underlined nucleotides are the start codon;SEQ ID NO:16). In other embodiments, restriction sites can be added orremoved to facilitate the cloning process.

Heterologous Nucleotide Sequences

In some embodiments, the isolated nucleic acid molecules of theinvention further comprise a heterologous nucleotide sequence. In someembodiments, the isolated nucleic acid molecules of the inventionfurther comprise at least one heterologous nucleotide sequence. Theheterologous nucleotide sequence can be linked with the optimizedBDD-FVIII nucleotide sequences of the invention at the 5′ end, at the 3′end, or inserted into the middle of the optimized BDD-FVIII nucleotidesequence. Thus, in some embodiments, the heterologous amino acidsequence encoded by the heterologous nucleotide sequence is linked tothe N-terminus or the C-terminus of the FVIII amino acid sequenceencoded by the nucleotide sequence or inserted between two amino acidsin the FVIII amino acid sequence. In other embodiments, the isolatednucleic acid molecules of the invention further comprise two, three,four, five, six, seven, or eight heterologous nucleotide sequences. Insome embodiments, all the heterologous nucleotide sequences areidentical. In some embodiments, at least one heterologous nucleotidesequence is different from the other heterologous nucleotide sequences.In some embodiments, the invention can comprise two, three, four, five,six, or more than seven heterologous nucleotide sequences in tandem.

In some embodiments, the heterologous nucleotide sequence encodes anamino acid sequence. In some embodiments, the amino acid sequenceencoded by the heterologous nucleotide sequence is a heterologous moietythat can increase the half-life (a “half-life extender”) of a FVIIImolecule.

In some embodiments, the heterologous moiety is a peptide or apolypeptide with either unstructured or structured characteristics thatare associated with the prolongation of in vivo half-life whenincorporated in a protein of the invention. Non-limiting examplesinclude albumin, albumin fragments, Fc fragments of immunoglobulins, theC-terminal peptide (CTP) of the β subunit of human chorionicgonadotropin, a HAP sequence, an XTEN sequence, a transferrin or afragment thereof, a PAS polypeptide, polyglycine linkers, polyserinelinkers, albumin-binding moieties, or any fragments, derivatives,variants, or combinations of these polypeptides. In some aspects, aheterologous moiety includes von Willebrand factor or a fragmentthereof. In other related aspects a heterologous moiety can include anattachment site (e.g., a cysteine amino acid) for a non-polypeptidemoiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES),polysialic acid, or any derivatives, variants, or combinations of theseelements. In some aspects, a heterologous moiety comprises a cysteineamino acid that functions as an attachment site for a non-polypeptidemoiety such as polyethylene glycol (PEG), hydroxyethyl starch (HES),polysialic acid, or any derivatives, variants, or combinations of theseelements.

In one specific embodiment, a first heterologous nucleotide sequenceencodes a first heterologous moiety that is a half-life extendingmolecule which is known in the art, and a second heterologous nucleotidesequence encodes a second heterologous moiety that can also be ahalf-life extending molecule which is known in the art. In certainembodiments, the first heterologous moiety (e.g., a first Fc moiety) andthe second heterologous moiety (e.g., a second Fc moiety) are associatedwith each other to form a dimer. In one embodiment, the secondheterologous moiety is a second Fc moiety, wherein the second Fc moietyis linked to or associated with the first heterologous moiety, e.g., thefirst Fc moiety. For example, the second heterologous moiety (e.g., thesecond Fc moiety) can be linked to the first heterologous moiety (e.g.,the first Fc moiety) by a linker or associated with the firstheterologous moiety by a covalent or non-covalent bond.

In some embodiments, the heterologous moiety is a polypeptidecomprising, consisting essentially of, or consisting of at least about10, at least about 100, at least about 200, at least about 300, at leastabout 400, at least about 500, at least about 600, at least about 700,at least about 800, at least about 900, at least about 1000, at leastabout 1100, at least about 1200, at least about 1300, at least about1400, at least about 1500, at least about 1600, at least about 1700, atleast about 1800, at least about 1900, at least about 2000, at leastabout 2500, at least about 3000, or at least about 4000 amino acids. Inother embodiments, the heterologous moiety is a polypeptide comprising,consisting essentially of, or consisting of about 100 to about 200 aminoacids, about 200 to about 300 amino acids, about 300 to about 400 aminoacids, about 400 to about 500 amino acids, about 500 to about 600 aminoacids, about 600 to about 700 amino acids, about 700 to about 800 aminoacids, about 800 to about 900 amino acids, or about 900 to about 1000amino acids.

In certain embodiments, a heterologous moiety improves one or morepharmacokinetic properties of the FVIII protein without significantlyaffecting its biological activity or function.

In certain embodiments, a heterologous moiety increases the in vivoand/or in vitro half-life of the FVIII protein of the invention. Inother embodiments, a heterologous moiety facilitates visualization orlocalization of the FVIII protein of the invention or a fragment thereof(e.g., a fragment comprising a heterologous moiety after proteolyticcleavage of the FVIII protein). Visualization and/or location of theFVIII protein of the invention or a fragment thereof can be in vivo, invitro, ex vivo, or combinations thereof.

In other embodiments, a heterologous moiety increases stability of theFVIII protein of the invention or a fragment thereof (e.g., a fragmentcomprising a heterologous moiety after proteolytic cleavage of the FVIIIprotein). As used herein, the term “stability” refers to anart-recognized measure of the maintenance of one or more physicalproperties of the FVIII protein in response to an environmentalcondition (e.g., an elevated or lowered temperature). In certainaspects, the physical property can be the maintenance of the covalentstructure of the FVIII protein (e.g., the absence of proteolyticcleavage, unwanted oxidation or deamidation). In other aspects, thephysical property can also be the presence of the FVIII protein in aproperly folded state (e.g., the absence of soluble or insolubleaggregates or precipitates). In one aspect, the stability of the FVIIIprotein is measured by assaying a biophysical property of the FVIIIprotein, for example thermal stability, pH unfolding profile, stableremoval of glycosylation, solubility, biochemical function (e.g.,ability to bind to a protein, receptor or ligand), etc., and/orcombinations thereof. In another aspect, biochemical function isdemonstrated by the binding affinity of the interaction. In one aspect,a measure of protein stability is thermal stability, i.e., resistance tothermal challenge. Stability can be measured using methods known in theart, such as, HPLC (high performance liquid chromatography), SEC (sizeexclusion chromatography), DLS (dynamic light scattering), etc. Methodsto measure thermal stability include, but are not limited todifferential scanning calorimetry (DSC), differential scanningfluorimetry (DSF), circular dichroism (CD), and thermal challenge assay.

In certain aspects, a FVIII protein of the invention comprises at leastone half-life extender, i.e., a heterologous moiety which increases thein vivo half-life of the FVIII protein with respect to the in vivohalf-life of the corresponding FVIII protein lacking such heterologousmoiety. In vivo half-life of a FVIII protein can be determined by anymethods known to those of skill in the art, e.g., activity assays(chromogenic assay or one stage clotting aPTT assay), ELISA, ROTEM™,etc.

In some embodiments, the presence of one or more half-life extendersresults in the half-life of the FVIII protein to be increased comparedto the half-life of the corresponding protein lacking such one or morehalf-life extenders. The half-life of the FVIII protein comprising ahalf-life extender is at least about 1.5 times, at least about 2 times,at least about 2.5 times, at least about 3 times, at least about 4times, at least about 5 times, at least about 6 times, at least about 7times, at least about 8 times, at least about 9 times, at least about 10times, at least about 11 times, or at least about 12 times longer thanthe in vivo half-life of the corresponding FVIII protein lacking suchhalf-life extender.

In one embodiment, the half-life of the FVIII protein comprising ahalf-life extender is about 1.5-fold to about 20-fold, about 1.5 fold toabout 15 fold, or about 1.5 fold to about 10 fold longer than the invivo hall-life of the corresponding protein lacking such half-lifeextender. In another embodiment, the half-life of FVIII proteincomprising a half-life extender is extended about 2-fold to about10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold,about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-foldto about 5-fold, about 2-fold to about 4-fold, about 2-fold to about3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold,about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about2.5-fold to about 6-fold, about 2.5-fold to about 5-fold, about 2.5-foldto about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to about10-fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold,about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-foldto about 5-fold, about 3-fold to about 4-fold, about 4-fold to about 6fold, about 5-fold to about 7-fold, or about 6-fold to about 8 fold ascompared to the in vivo half-life of the corresponding protein lackingsuch half-life extender.

In other embodiments, the half-life of the FVIII protein comprising ahalf-life extender is at least about 17 hours, at least about 18 hours,at least about 19 hours, at least about 20 hours, at least about 21hours, at least about 22 hours, at least about 23 hours, at least about24 hours, at least about 25 hours, at least about 26 hours, at leastabout 27 hours, at least about 28 hours, at least about 29 hours, atleast about 30 hours, at least about 31 hours, at least about 32 hours,at least about 33 hours, at least about 34 hours, at least about 35hours, at least about 36 hours, at least about 48 hours, at least about60 hours, at least about 72 hours, at least about 84 hours, at leastabout 96 hours, or at least about 108 hours.

In still other embodiments, the half-life of the FVIII proteincomprising a half-life extender is about 15 hours to about two weeks,about 16 hours to about one week, about 17 hours to about one week,about 18 hours to about one week, about 19 hours to about one week,about 20 hours to about one week, about 21 hours to about one week,about 22 hours to about one week, about 23 hours to about one week,about 24 hours to about one week, about 36 hours to about one week,about 48 hours to about one week, about 60 hours to about one week,about 24 hours to about six days, about 24 hours to about five days,about 24 hours to about four days, about 24 hours to about three days,or about 24 hours to about two days.

In some embodiments, the average half-life per subject of the FVIIIprotein comprising a half-life extender is about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day),about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours,about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72hours (3 days), about 84 hours, about 96 hours (4 days), about 108hours, about 120 hours (5 days), about six days, about seven days (oneweek), about eight days, about nine days, about 10 days, about 11 days,about 12 days, about 13 days, or about 14 days.

1. An Immunoglobulin Constant Region or a Portion Thereof

In another aspect, a heterologous moiety comprises one or moreimmunoglobulin constant regions or portions thereof (e.g., an Fcregion). In one embodiment, an isolated nucleic acid molecule of theinvention further comprises a heterologous nucleic acid sequence thatencodes an immunoglobulin constant region or a portion thereof. In someembodiments, the immunoglobulin constant region or portion thereof is anFc region.

An immunoglobulin constant region is comprised of domains denoted CH(constant heavy) domains (CH1, CH2, etc.). Depending on the isotype,(i.e. IgG, IgM, IgA IgD, or IgE), the constant region can be comprisedof three or four CH domains. Some isotypes (e.g. IgG) constant regionsalso contain a hinge region. See Janeway et al. 2001, Immunobiology,Garland Publishing, N.Y., N.Y.

An immunoglobulin constant region or a portion thereof for producing theFVIII protein of the present invention can be obtained from a number ofdifferent sources. In one embodiment, an immunoglobulin constant regionor a portion thereof is derived from a human immunoglobulin. It isunderstood, however, that the immunoglobulin constant region or aportion thereof can be derived from an immunoglobulin of anothermammalian species, including for example, a rodent (e.g. a mouse, rat,rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque)species. Moreover, the immunoglobulin constant region or a portionthereof can be derived from any immunoglobulin class, including IgM,IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgG1,IgG2, IgG3 and IgG4. In one embodiment, the human isotype IgG1 is used.

A variety of the immunoglobulin constant region gene sequences (e.g.human constant region gene sequences) are available in the form ofpublicly accessible deposits. Constant region domains sequence can beselected having a particular effector function (or lacking a particulareffector function) or with a particular modification to reduceimmunogenicity. Many sequences of antibodies and antibody-encoding geneshave been published and suitable Ig constant region sequences (e.g.hinge, CH2, and/or CH3 sequences, or portions thereof) can be derivedfrom these sequences using art recognized techniques. The geneticmaterial obtained using any of the foregoing methods can then be alteredor synthesized to obtain polypeptides of the present invention. It willfurther be appreciated that the scope of this invention encompassesalleles, variants and mutations of constant region DNA sequences.

The sequences of the immunoglobulin constant region or a portion thereofcan be cloned, e.g., using the polymerase chain reaction and primerswhich are selected to amplify the domain of interest. To clone asequence of the immunoglobulin constant region or a portion thereof froman antibody, mRNA can be isolated from hybridoma, spleen, or lymphcells, reverse transcribed into DNA, and antibody genes amplified byPCR. PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., “PCRProtocols: A Guide to Methods and Applications” Innis et al. eds.,Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51;Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated byconsensus constant region primers or by more specific primers based onthe published heavy and light chain DNA and amino acid sequences. PCRalso can be used to isolate DNA clones encoding the antibody light andheavy chains. In this case the libraries can be screened by consensusprimers or larger homologous probes, such as mouse constant regionprobes. Numerous primer sets suitable for amplification of antibodygenes are known in the art (e.g., 5′ primers based on the N-terminalsequence of purified antibodies (Benhar and Pastan. 1994. ProteinEngineering 7:1509); rapid amplification of eDNA ends (Ruberti, F. etal. 1994. J Immunol. Methods 173:33); antibody leader sequences (Larricket al, 1989 Biochem. Biophys. Res. Commun. 160:1250). The cloning ofantibody sequences is further described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein.

An immunoglobulin constant region used herein can include all domainsand the hinge region or portions thereof. In one embodiment, theimmunoglobulin constant region or a portion thereof comprises CH2domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRnbinding partner.

As used herein, the term “Fc region” is defined as the portion of apolypeptide which corresponds to the Fc region of native Ig, i.e., asformed by the dimeric association of the respective Fc domains of itstwo heavy chains. A native Fc region forms a homodimer with another Fcregion. In contrast, the term “genetically-fused Fc region” or“single-chain Fc region” (scFc region), as used herein, refers to asynthetic dimeric Fc region comprised of Fc domains genetically linkedwithin a single polypeptide chain (i.e., encoded in a single contiguousgenetic sequence). See International Publication No. WO 2012/006635,incorporated herein by reference in its entirety.

In one embodiment, the “Fc region” refers to the portion of a single Igheavy chain beginning in the hinge region just upstream of the papaincleavage site (i.e. residue 216 in IgG, taking the first residue ofheavy chain constant region to be 114) and ending at the C-terminus ofthe antibody. Accordingly, a complete Fc region comprises at least ahinge domain, a CH2 domain, and a CH3 domain.

An immunoglobulin constant region or a portion thereof can be an FcRnbinding partner. FcRn is active in adult epithelial tissues andexpressed in the lumen of the intestines, pulmonary airways, nasalsurfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No.6,485,726). An FcRn binding partner is a portion of an immunoglobulinthat binds to FcRn.

The FcRn receptor has been isolated from several mammalian speciesincluding humans. The sequences of the human FcRn, monkey FcRn, ratFcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med.180:2377). The FcRn receptor binds IgG (but not other immunoglobulinclasses such as IgA, IgM, IgD, and IgE) at relatively low pH, activelytransports the IgG transcellularly in a luminal to serosal direction,and then releases the IgG at relatively higher pH found in theinterstitial fluids. It is expressed in adult epithelial tissue (U.S.Pat. Nos. 6,485,726, 6,030,613, 6,086,875; WO 03/077834;US2003-0235536A1) including lung and intestinal epithelium (Israel etal. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashiet al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasalepithelium, vaginal surfaces, and biliary tree surfaces.

FcRn binding partners useful in the present invention encompassmolecules that can be specifically bound by the FcRn receptor includingwhole IgG, the Fc fragment of IgG, and other fragments that include thecomplete binding region of the FcRn receptor. The region of the Fcportion of IgG that binds to the FcRn receptor has been described basedon X-ray crystallography (Burmeister et al. 1994, Nature 372:379). Themajor contact area of the Fc with the FcRn is near the junction of theCH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavychain. The FcRn binding partners include whole IgG, the Fc fragment ofIgG, and other fragments of IgG that include the complete binding regionof FcRn. The major contact sites include amino acid residues 248,250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain andamino acid residues 385-387, 428, and 433-436 of the CH3 domain.References made to amino acid numbering of immunoglobulins orimmunoglobulin fragments, or regions, are all based on Kabat et al.1991, Sequences of Proteins of Immunological Interest, U.S. Departmentof Public Health, Bethesda, Md.

Fc regions or FcRn binding partners bound to FcRn can be effectivelyshuttled across epithelial barriers by FcRn, thus providing anon-invasive means to systemically administer a desired therapeuticmolecule. Additionally, fusion proteins comprising an Fc region or anFcRn binding partner are endocytosed by cells expressing the FcRn. Butinstead of being marked for degradation, these fusion proteins arerecycled out into circulation again, thus increasing the in vivohalf-life of these proteins. In certain embodiments, the portions ofimmunoglobulin constant regions are an Fc region or an FcRn bindingpartner that typically associates, via disulfide bonds and othernon-specific interactions, with another Fc region or another FcRnbinding partner to form dimers and higher order multimers.

Two FcRn receptors can bind a single Fc molecule. Crystallographic datasuggest that each FcRn molecule binds a single polypeptide of the Fchomodimer. In one embodiment, linking the FcRn binding partner, e.g., anFc fragment of an IgG, to a biologically active molecule provides ameans of delivering the biologically active molecule orally, buccally,sublingually, rectally, vaginally, as an aerosol administered nasally orvia a pulmonary route, or via an ocular route. In another embodiment,the FVIII protein can be administered invasively, e.g., subcutaneously,intravenously.

An FcRn binding partner region is a molecule or portion thereof that canbe specifically bound by the FcRn receptor with consequent activetransport by the FcRn receptor of the Fc region. Specifically boundrefers to two molecules forming a complex that is relatively stableunder physiologic conditions. Specific binding is characterized by ahigh affinity and a low to moderate capacity as distinguished fromnonspecific binding which usually has a low affinity with a moderate tohigh capacity. Typically, binding is considered specific when theaffinity constant KA is higher than 10⁶ M⁻¹, or higher than 10⁸ M⁻¹. Ifnecessary, non-specific binding can be reduced without substantiallyaffecting specific binding by varying the binding conditions. Theappropriate binding conditions such as concentration of the molecules,ionic strength of the solution, temperature, time allowed for binding,concentration of a blocking agent (e.g., serum albumin, milk casein),etc., can be optimized by a skilled artisan using routine techniques.

In certain embodiments, a FVIII protein of the invention comprises oneor more truncated Fc regions that are nonetheless sufficient to conferFc receptor (FcR) binding properties to the Fc region. For example, theportion of an Fc region that binds to FcRn (i.e., the FcRn bindingportion) comprises from about amino acids 282-438 of IgG, EU numbering(with the primary contact sites being amino acids 248, 250-257, 272,285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acidresidues 385-387, 428, and 433-436 of the CH3 domain. Thus, an Fc regionof the invention can comprise or consist of an FcRn binding portion.FcRn binding portions can be derived from heavy chains of any isotype,including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn bindingportion from an antibody of the human isotype IgG1 is used. In anotherembodiment, an FcRn binding portion from an antibody of the humanisotype IgG4 is used.

The Fc region can be obtained from a number of different sources. In oneembodiment, an Fc region of the polypeptide is derived from a humanimmunoglobulin. It is understood, however, that an Fc moiety can bederived from an immunoglobulin of another mammalian species, includingfor example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) ornon-human primate (e.g. chimpanzee, macaque) species. Moreover, thepolypeptide of the Fc domains or portions thereof can be derived fromany immunoglobulin class, including IgM, IgG, IgD, IgA and IgE, and anyimmunoglobulin isotype, including IgG1, IgG2, IgG3 and IgG4. In anotherembodiment, the human isotype IgG is used.

In certain embodiments, the Fc variant confers a change in at least oneeffector function imparted by an Fc moiety comprising said wild-type Fcdomain (e.g., an improvement or reduction in the ability of the Fcregion to bind to Fc receptors (e.g. FcγRI, FcγRII, or FcγRIII) orcomplement proteins (e.g. C1q), or to trigger antibody-dependentcytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity(CDCC)). In other embodiments, the Fc variant provides an engineeredcysteine residue.

The Fc region of the invention can employ art-recognized Fc variantswhich are known to impart a change (e.g., an enhancement or reduction)in effector function and/or FcR or FcRn binding. Specifically, an Fcregion of the invention can include, for example, a change (e.g., asubstitution) at one or more of the amino acid positions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO04/044859, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2; US Patent Publication Nos. US2007/0231329,US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767,US2007/0243188, US20070248603, US20070286859, US20080057056; or U.S.Pat. Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871;6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124;6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091,each of which is incorporated by reference herein. In one embodiment,the specific change (e.g., the specific substitution of one or moreamino acids disclosed in the art) can be made at one or more of thedisclosed amino acid positions. In another embodiment, a differentchange at one or more of the disclosed amino acid positions (e.g., thedifferent substitution of one or more amino acid position disclosed inthe art) can be made.

The Fc region or FcRn binding partner of IgG can be modified accordingto well recognized procedures such as site directed mutagenesis and thelike to yield modified IgG or Fc fragments or portions thereof that willbe bound by FcRn. Such modifications include modifications remote fromthe FcRn contact sites as well as modifications within the contact sitesthat preserve or even enhance binding to the FcRn. For example, thefollowing single amino acid residues in human IgG1 Fc (Fc γ1) can besubstituted without significant loss of Fc binding affinity for FcRn:P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A,S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A,E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F,N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A,N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q,P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A,Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A,D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A,Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A,N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A,and K447A, where for example P238A represents wild type prolinesubstituted by alanine at position number 238. As an example, a specificembodiment incorporates the N297A mutation, removing a highly conservedN-glycosylation site. In addition to alanine other amino acids can besubstituted for the wild type amino acids at the positions specifiedabove. Mutations can be introduced singly into Fc giving rise to morethan one hundred Fc regions distinct from the native Fc. Additionally,combinations of two, three, or more of these individual mutations can beintroduced together, giving rise to hundreds more Fc regions.

Certain of the above mutations can confer new functionality upon the Fcregion or FcRn binding partner. For example, one embodiment incorporatesN297A, removing a highly conserved N-glycosylation site. The effect ofthis mutation is to reduce immunogenicity, thereby enhancing circulatinghalf-life of the Fc region, and to render the Fc region incapable ofbinding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromisingaffinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friendet al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol.Chem. 276:6591). As a further example of new functionality arising frommutations described above affinity for FcRn can be increased beyond thatof wild type in some instances. This increased affinity can reflect anincreased “on” rate, a decreased “off” rate or both an increased “on”rate and a decreased “off” rate. Examples of mutations believed toimpart an increased affinity for FcRn include, but not limited to,T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem.276:6591).

Additionally, at least three human Fc gamma receptors appear torecognize a binding site on IgG within the lower hinge region, generallyamino acids 234-237. Therefore, another example of new functionality andpotential decreased immunogenicity can arise from mutations of thisregion, as for example by replacing amino acids 233-236 of human IgG1“ELLG” (SEQ ID NO:29) to the corresponding sequence from IgG2 “PVA”(with one amino acid deletion). It has been shown that FcγRI, FcγRII,and FcγRIII, which mediate various effector functions will not bind toIgG1 when such mutations have been introduced. Ward and Ghetie 1995,Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol.29:2613.

In another embodiment, the immunoglobulin constant region or a portionthereof comprises an amino acid sequence in the hinge region or aportion thereof that forms one or more disulfide bonds with a secondimmunoglobulin constant region or a portion thereof. The secondimmunoglobulin constant region or a portion thereof an be linked to asecond polypeptide, bringing the FVIII protein and the secondpolypeptide together. In some embodiments, the second polypeptide is anenhancer moiety. As used herein, the term “enhancer moiety” refers to amolecule, fragment thereof or a component of a polypeptide which iscapable of enhancing the procoagulant activity of FVIII. The enhancermoiety can be a cofactor, such as soluble tissue factor (sTF), or aprocoagulant peptide. Thus, upon activation of FVIII, the enhancermoiety is available to enhance FVIII activity.

In certain embodiments, a FVIII protein of the invention comprises anamino acid substitution to an immunoglobulin constant region or aportion thereof (e.g., Fc variants), which alters theantigen-independent effector functions of the Ig constant region, inparticular the circulating half-life of the protein.

2. scFc Regions

In another aspect, a heterologous moiety comprises a scFc (single chainFc) region. In one embodiment, an isolated nucleic acid molecule of theinvention further comprises a heterologous nucleic acid sequence thatencodes a scFc region. The scFc region comprises at least twoimmunoglobulin constant regions or portions thereof (e.g., Fc moietiesor domains (e.g., 2, 3, 4, 5, 6, or more Fc moieties or domains)) withinthe same linear polypeptide chain that are capable of folding (e.g.,intramolecularly or intermolecularly folding) to form one functionalscFc region which is linked by an Fc peptide linker. For example, in oneembodiment, a polypeptide of the invention is capable of binding, viaits scFc region, to at least one Fc receptor (e.g. an FcRn, an FcγRreceptor (e.g., FcγRIII), or a complement protein (e.g. C1q)) in orderto improve half-life or trigger an immune effector function (e.g.,antibody-dependent cytotoxicity (ADCC), phagocytosis, orcomplement-dependent cytotoxicity (CDCC) and/or to improvemanufacturability).

3. CTP

In another aspect, a heterologous moiety comprises one C-terminalpeptide (CTP) of the β subunit of human chorionic gonadotropin orfragment, variant, or derivative thereof, One or more CTP peptidesinserted into a recombinant protein is known to increase the in vivohalf-life of that protein. See, e.g., U.S. Pat. No. 5,712,122,incorporated by reference herein in its entirety.

Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ IDNO:17) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ. (SEQ ID NO:18). See, e.g., U.S.Patent Application Publication No. US 2009/0087411 A1, incorporated byreference.

4. XTEN Sequence

In some embodiments, a heterologous moiety comprises one or more XTENsequences, fragments, variants, or derivatives thereof. As used here“XTEN sequence” refers to extended length polypeptides withnon-naturally occurring, substantially non-repetitive sequences that arecomposed mainly of small hydrophilic amino acids, with the sequencehaving a low degree or no secondary or tertiary structure underphysiologic conditions. As a heterologous moiety, XTENs can serve as ahalf-life extension moiety. In addition, XTEN can provide desirableproperties including but are not limited to enhanced pharmacokineticparameters and solubility characteristics.

The incorporation of a heterologous moiety comprising an XTEN sequenceinto a protein of the invention can confer to the protein one or more ofthe following advantageous properties: conformational flexibility,enhanced aqueous solubility, high degree of protease resistance, lowimmunogenicity, low binding to mammalian receptors, or increasedhydrodynamic (or Stokes) radii.

In certain aspects, an XTEN sequence can increase pharmacokineticproperties such as longer in vivo half-life or increased area under thecurve (AUC), so that a protein of the invention stays in vivo and hasprocoagulant activity for an increased period of time compared to aprotein with the same but without the XTEN heterologous moiety.

Examples of XTEN sequences that can be used as heterologous moieties inchimeric proteins of the invention are disclosed, e.g., in U.S. PatentPublication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1,2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or InternationalPatent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, or WO 2011028344 A2,each of which is incorporated by reference herein in its entirety.

Exemplary XTEN sequences that can be used as heterologous moieties inchimeric protein of the invention include XTEN AE42-4 (SEQ ID NO:30,encoded by SEQ ID NO:31), XTEN 144-2A (SEQ ID NO:32, encoded by SEQ IDNO:33), XTEN A144-3B (SEQ ID NO:34, encoded by SEQ ID NO:35), XTENAE144-4A (SEQ ID NO:36, encoded by SEQ ID NO:37), XTEN AE144-5A (SEQ IDNO:38, encoded by SEQ ID NO:39), XTEN AE144-6B (SEQ ID NO:40, encoded bySEQ ID NO:41), XTEN AG144-1 (SEQ ID NO:42, encoded by SEQ ID NO:43),XTEN AG144-A (SEQ ID NO:44, encoded by SEQ ID NO:45), XTEN AG144-B (SEQID NO:46, encoded by SEQ ID NO:47), XTEN AG144-C(SEQ ID NO:48, encodedby SEQ ID NO:49), and XTEN AG144-F (SEQ ID NO:50, encoded by SEQ IDNO:51).

5. Albumin or Fragment, Derivative, or Variant Thereof

In some embodiments, a heterologous moiety comprises albumin or afunctional fragment thereof. Human serum albumin (HSA, or HA), a proteinof 609 amino acids in its full-length form, is responsible for asignificant proportion of the osmotic pressure of serum and alsofunctions as a carrier of endogenous and exogenous ligands. The term“albumin” as used herein includes full-length albumin or a functionalfragment, variant, derivative, or analog thereof. Examples of albumin orthe fragments or variants thereof are disclosed in US Pat. Publ. Nos.2008/0194481A1, 2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or2008/0153751 A1 or PCT Appl. Publ. Nos. 2008/033413 A2, 2009/058322 A1,or 2007/021494 A2, which are incorporated herein by reference in theirentireties.

In one embodiment, the FVIII protein of the invention comprises albumin,a fragment, or a variant thereof which is further linked to a secondheterologous moiety selected from the group consisting of animmunoglobulin constant region or portion thereof (e.g., an Fc region),a PAS sequence, HES, and PEG.

6. Albumin-Binding Moiety

In certain embodiments, the heterologous moiety is an albumin-bindingmoiety, which comprises an albumin-binding peptide, a bacterialalbumin-binding domain, an albumin-binding antibody fragment, or anycombinations thereof.

For example, the albumin-binding protein can be a bacterialalbumin-binding protein, an antibody or an antibody fragment includingdomain antibodies (see U.S. Pat. No. 6,696,245). An albumin-bindingprotein, for example, can be a bacterial albumin-binding domain, such asthe one of streptococcal protein G (Konig, T. and Skerra, A. (11998) J.Immunol. Methods 218, 73-83). Other examples of albumin-binding peptidesthat can be used as conjugation partner are, for instance, those havinga Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, H is, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al.(Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

Domain 3 from streptococcal protein G, as disclosed by Kraulis et al.,FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci.11:206-213 (2002) is an example of a bacterial albumin-binding domain.Examples of albumin-binding peptides include a series of peptides havingthe core sequence DICLPRWGCLW (SEQ ID NO:19). See, e.g., Dennis et al.,J. Biol. Chem. 2002, 277: 35035-35043 (2002). Examples ofalbumin-binding antibody fragments are disclosed in Muller andKontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al.,Cancer Immunol. Immunother. 56:303-317 (2007), and Holt et al., Prot.Eng. Design Sci., 21:283-288 (2008), which are incorporated herein byreference in their entireties. An example of such albumin-binding moietyis 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido) hexanoate(“Albu” tag) as disclosed by Trussel et al., Bioconjugate Chem.20:2286-2292 (2009).

Fatty acids, in particular long chain fatty acids (LCFA) and long chainfatty acid-like albumin-binding compounds can be used to extend the invivo half-life of FVIII proteins of the invention. An example of aLCFA-like albumin-binding compound is16-(1-(3-(9-(((2,5-dioxopyrrolidin-1-yloxy)carbonyloxy)-methyi)-7-sulfo-9H-fluoren-2-ylamino)-3-oxopropyl)-2,5-dioxopyrrolidin-3-ylthio)hexadecanoic acid (see, e.g., WO 2010/140148).

7. PAS Sequence

In other embodiments, the heterologous moiety is a PAS sequence. A PASsequence, as used herein, means an amino acid sequence comprising mainlyalanine and serine residues or comprising mainly alanine, serine, andproline residues, the amino acid sequence forming random coilconformation under physiological conditions. Accordingly, the PASsequence is a building block, an amino acid polymer, or a sequencecassette comprising, consisting essentially of, or consisting ofalanine, serine, and proline which can be used as a part of theheterologous moiety in the chimeric protein. Yet, the skilled person isaware that an amino acid polymer also can form random coil conformationwhen residues other than alanine, serine, and proline are added as aminor constituent in the PAS sequence. The term “minor constituent” asused herein means that amino acids other than alanine, serine, andproline can be added in the PAS sequence to a certain degree, e.g., upto about 12%, i.e., about 12 of 100 amino acids of the PAS sequence, upto about 10%, i.e. about 10 of 100 amino acids of the PAS sequence, upto about 9%, i.e., about 9 of 100 amino acids, up to about 8%, i.e.,about 8 of 100 amino acids, about 6%, i.e., about 6 of 100 amino acids,about 5%, i.e., about 5 of 100 amino acids, about 4%, i.e., about 4 of100 amino acids, about 3%, i.e., about 3 of 100 amino acids, about 2%,i.e., about 2 of 100 amino acids, about 1%, i.e., about 1 of 100 of theamino acids. The amino acids different from alanine, serine and prolinecan be selected from the group consisting of Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val.

Under physiological conditions, the PAS sequence stretch forms a randomcoil conformation and thereby can mediate an increased in vivo and/or invitro stability to the FVIII protein. Since the random coil domain doesnot adopt a stable structure or function by itself, the biologicalactivity mediated by the FVIII protein is essentially preserved. Inother embodiments, the PAS sequences that form random coil domain arebiologically inert, especially with respect to proteolysis in bloodplasma, immunogenicity, isoelectric point/electrostatic behaviour,binding to cell surface receptors or internalisation, but are stillbiodegradable, which provides clear advantages over synthetic polymerssuch as PEG.

Non-limiting examples of the PAS sequences forming random coilconformation comprise an amino acid sequence selected from the groupconsisting of ASPAAPASPAAPAPSAPA (SEQ ID NO: 20), AAPASPAPAAPSAPAPAAPS(SEQ ID NO: 21), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 22),APSSPSPSAPSSPSPASPS (SEQ ID NO: 23), SSPSAPSPSSPASPSPSSPA (SEQ ID NO:24), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 25) and ASAAAPAAASAAASAPSAAA(SEQ ID NO: 26) or any combinations thereof. Additional examples of PASsequences are known from, e.g., US Pat. Publ. No. 2010/0292130 A1 andPCT Appl. Publ. No. WO 2008/155134 A1.

8. HAP Sequence

In certain embodiments, the heterologous moiety is a glycine-richhomo-amino-acid polymer (HAP). The HAP sequence can comprise arepetitive sequence of glycine, which has at least 50 amino acids, atleast 100 amino acids, 120 amino acids, 140 amino acids, 160 aminoacids, 180 amino acids, 200 amino acids, 250 amino acids, 300 aminoacids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 aminoacids in length. In one embodiment, the HAP sequence is capable ofextending half-life of a moiety fused to or linked to the HAP sequence.Non-limiting examples of the HAP sequence includes, but are not limitedto (Gly)_(n), (Gly₄Ser)_(n) or S(Gly₄Ser)_(n), wherein n is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In oneembodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.

9. Transferrin or Fragment Thereof

In certain embodiments, the heterologous moiety is transferrin or afragment thereof. Any transferrin can be used to make the FVIII proteinsof the invention. As an example, wild-type human TF (TF) is a 679 aminoacid protein, of approximately 75 KDa (not accounting forglycosylation), with two main domains, N (about 330 amino acids) and C(about 340 amino acids), which appear to originate from a geneduplication. See GenBank accession numbers NM001063, XM002793, M12530,XM039845, XM 039847 and S95936 (www.ncbi.nlm.nih.gov/), all of which areherein incorporated by reference in their entirety. Transferrincomprises two domains, N domain and C domain. N domain comprises twosubdomains, N1 domain and N2 domain, and C domain comprises twosubdomains, C1 domain and C2 domain.

In one embodiment, the transferrin heterologous moiety includes atransferrin splice variant. In one example, a transferrin splice variantcan be a splice variant of human transferrin, e.g., Genbank AccessionAAA61140. In another embodiment, the transferrin portion of the chimericprotein includes one or more domains of the transferrin sequence, e.g.,N domain, C domain, N1 domain, N2 domain, C1 domain, C2 domain or anycombinations thereof.

10. Clearance Receptors

In certain embodiments, the heterologous moiety is a clearance receptor,fragment, variant, or derivative thereof. LRP1 is a 600 kDa integralmembrane protein that is implicated in the receptor-mediate clearance ofa variety of proteins, such as Factor X. See, e.g., Narita et al., Blood91:555-560 (1998).

11. von Willebrand Factor or Fragments Thereof

In certain embodiments, the heterologous moiety is von Willebrand Factor(VWF) or fragments thereof.

VWF (also known as F8VWF) is a large multimeric glycoprotein present inblood plasma and produced constitutively in endothelium (in theWeibel-Palade bodies), megakaryocytes (α-granules of platelets), andsubendothelian connective tissue. The basic VWF monomer is a 2813 aminoacid protein. Every monomer contains a number of specific domains with aspecific function, the D′ and D3 domains (which together bind to FactorVIII), the A1 domain (which binds to platelet GPIb-receptor, heparin,and/or possibly collagen), the A3 domain (which binds to collagen), theC1 domain (in which the RGD domain binds to platelet integrin αIIbβ3when this is activated), and the “cysteine knot” domain at theC-terminal end of the protein (which VWF shares with platelet-derivedgrowth factor (PDGF), transforming growth factor-β (TGFβ) and β-humanchorionic gonadotropin (βHCG)).

The 2813 monomer amino acid sequence for human VWF is reported asAccession Number NP000543.2 in Genbank. The nucleotide sequence encodingthe human VWF is reported as Accession Number NM000552.3 in Genbank. Thenucleotide sequence of human VWF is designated as SEQ ID NO: 27. SEQ IDNO: 28 is the amino acid sequence encoded by SEQ ID NO: 27. The D′domain includes amino acids 764 to 866 of SEQ ID NO:28. The D3 domainincludes amino acids 867 to 1240 of SEQ ID NO:28.

In plasma, 95-98% of FVIII circulates in a tight non-covalent complexwith full-length VWF. The formation of this complex is important for themaintenance of appropriate plasma levels of FVIIII in vivo. Lenting etal., Blood 92(11): 3983-96 (1998); Lenting et al., J Thromb. Haemost.5(7): 1353-60 (2007). When FVIII is activated due to proteolysis atpositions 372 and 740 in the heavy chain and at position 1689 in thelight chain, the VWF bound to FVIII is removed from the activated FVIII.

In certain embodiments, the heterologous moiety is full length vonWillebrand Factor. In other embodiments, the heterologous moiety is avon Willebrand Factor fragment. As used herein, the term “VWF fragment”or “VWF fragments” used herein means any VWF fragments that interactwith FVIII and retain at least one or more properties that are normallyprovided to FVIII by full-length VWF, e.g., preventing prematureactivation to FVIIIa, preventing premature proteolysis, preventingassociation with phospholipid membranes that could lead to prematureclearance, preventing binding to FVIII clearance receptors that can bindnaked FVIII but not VWF-bound FVIII, and/or stabilizing the FVIII heavychain and light chain interactions. In a specific embodiment, theheterologous moiety is a (VWF) fragment comprising a D′ domain and a D3domain of VWF. The VWF fragment comprising the D′ domain and the D3domain can further comprise a VWF domain selected from the groupconsisting of an A1 domain, an A2 domain, an A3 domain, a D1 domain, aD2 domain, a D4 domain, a B1 domain, a B2 domain, a B3 domain, a C1domain, a C2 domain, a CK domain, one or more fragments thereof, and anycombinations thereof. Additional examples of the polypeptide havingFVIII activity fused to the VWF fragment are disclosed in U.S.provisional patent application No. 61/667,901, filed Jul. 3, 2012,incorporated herein by reference in its entirety.

12. Linker Moieties

In certain embodiments, the heterologous moiety is a peptide linker.

As used herein, the terms “peptide linkers” or “linker moieties” referto a peptide or polypeptide sequence (e.g., a synthetic peptide orpolypeptide sequence) which connects two domains in a linear amino acidsequence of a polypeptide chain.

In some embodiments, heterologous nucleotide sequences encoding peptidelinkers can be inserted between the optimized FVIII polynucleotidesequences of the invention and a heterologous nucleotide sequenceencoding, for example, one of the heterologous moieties described above,such as albumin. Peptide linkers can provide flexibility to the chimericpolypeptide molecule. Linkers are not typically cleaved, however suchcleavage can be desirable. In one embodiment, these linkers are notremoved during processing.

A type of linker which can be present in a chimeric protein of theinvention is a protease cleavable linker which comprises a cleavage site(i.e., a protease cleavage site substrate, e.g., a factor XIa, Xa, orthrombin cleavage site) and which can include additional linkers oneither the N-terminal of C-terminal or both sides of the cleavage site.These cleavable linkers when incorporated into a construct of theinvention result in a chimeric molecule having a heterologous cleavagesite.

In one embodiment, an FVIII polypeptide of the instant inventioncomprises two or more Fc domains or moieties linked via a cscFc linkerto form an Fc region comprised in a single polypeptide chain. The cscFclinker is flanked by at least one intracellular processing site, i.e., asite cleaved by an intracellular enzyme. Cleavage of the polypeptide atthe at least one intracellular processing site results in a polypeptidewhich comprises at least two polypeptide chains.

Other peptide linkers can optionally be used in a construct of theinvention, e.g., to connect an FVIII protein to an Fc region. Someexemplary linkers that can be used in connection with the inventioninclude, e.g., polypeptides comprising GlySer amino acids described inmore detail below.

In one embodiment, the peptide linker is synthetic, i.e., non-naturallyoccurring. In one embodiment, a peptide linker includes peptides (orpolypeptides) (which can or can not be naturally occurring) whichcomprise an amino acid sequence that links or genetically fuses a firstlinear sequence of amino acids to a second linear sequence of aminoacids to which it is not naturally linked or genetically fused innature. For example, in one embodiment the peptide linker can comprisenon-naturally occurring polypeptides which are modified forms ofnaturally occurring polypeptides (e.g., comprising a mutation such as anaddition, substitution or deletion). In another embodiment, the peptidelinker can comprise non-naturally occurring amino acids. In anotherembodiment, the peptide linker can comprise naturally occurring aminoacids occurring in a linear sequence that does not occur in nature. Instill another embodiment, the peptide linker can comprise a naturallyoccurring polypeptide sequence.

For example, in certain embodiments, a peptide linker can be used tofuse identical Fc moieties, thereby forming a homodimeric scFc region.In other embodiments, a peptide linker can be used to fuse different Fcmoieties (e.g. a wild-type Fc moiety and an Fc moiety variant), therebyforming a heterodimeric scFc region.

In another embodiment, a peptide linker comprises or consists of agly-ser linker. In one embodiment, a scFc or cscFc linker comprises atleast a portion of an immunoglobulin hinge and a gly-ser linker. As usedherein, the term “gly-ser linker” refers to a peptide that consists ofglycine and serine residues. In certain embodiments, said gly-ser linkercan be inserted between two other sequences of the peptide linker. Inother embodiments, a gly-ser linker is attached at one or both ends ofanother sequence of the peptide linker. In yet other embodiments, two ormore gly-ser linker are incorporated in series in a peptide linker. Inone embodiment, a peptide linker of the invention comprises at least aportion of an upper hinge region (e.g., derived from an IgG1, IgG2,IgG3, or IgG4 molecule), at least a portion of a middle hinge region(e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a seriesof gly/ser amino acid residues.

Peptide linkers of the invention are at least one amino acid in lengthand can be of varying lengths. In one embodiment, a peptide linker ofthe invention is from about 1 to about 50 amino acids in length. As usedin this context, the term “about” indicates +/− two amino acid residues.Since linker length must be a positive interger, the length of fromabout 1 to about 50 amino acids in length, means a length of from 1-3 to48-52 amino acids in length. In another embodiment, a peptide linker ofthe invention is from about 10 to about 20 amino acids in length. Inanother embodiment, a peptide linker of the invention is from about 15to about 50 amino acids in length. In another embodiment, a peptidelinker of the invention is from about 20 to about 45 amino acids inlength. In another embodiment, a peptide linker of the invention is fromabout 15 to about 35 or about 20 to about 30 amino acids in length. Inanother embodiment, a peptide linker of the invention is from about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 500,1000, or 2000 amino acids in length. In one embodiment, a peptide linkerof the invention is 20 or 30 amino acids in length.

In some embodiments, the peptide linker can comprise at least two, atleast three, at least four, at least five, at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, or at least 100 amino acids. In other embodiments, thepeptide linker can comprise at least 200, at least 300, at least 400, atleast 500, at least 600, at least 700, at least 800, at least 900, or atleast 1,000 amino acids. In some embodiments, the peptide linker cancomprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The peptide linkercan comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 aminoacids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids,600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or900-1000 amino acids.

Peptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

Monomer-Dimer Hybrids

In some embodiments, the isolated nucleic acid molecules of theinvention which farther comprise a heterologous nucleotide sequenceencode a monomer-dimer hybrid molecule comprising FVIII.

The term “monomer-dimer hybrid” used herein refers to a chimeric proteincomprising a first polypeptide chain and a second polypeptide chain,which are associated with each other by a disulfide bond, wherein thefirst chain comprises Factor VIII and a first Fc region and the secondchain comprises, consists essentially of, or consists of a second Fcregion without the FVIII. The monomer-dimer hybrid construct thus is ahybrid comprising a monomer aspect having only one clotting factor and adimer aspect having two Fc regions.

Transcription Control Sequences

In some embodiments, the isolated nucleic acid molecules of theinvention are operatively linked to at least one transcription controlsequences. A transcription control sequences as used herein is anyregulatory nucleotide sequence, such as a promoter sequence orpromoter-enhancer combination, which facilitates the efficienttranscription and translation of the coding nucleic acid to which it isoperably linked. The gene expression control sequence can, for example,be a mammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the transcription control sequences shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined coding nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

Vectors

The invention also provides vectors comprising the isolated nucleic acidmolecules of the invention. Suitable vectors include expression vectors,viral vectors, and plasmid vectors.

As used herein, an expression vector refers to any nucleic acidconstruct which contains the necessary elements for the transcriptionand translation of an inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation,when introduced into an appropriate host cell. Expression vectors caninclude plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of the invention will include optimizedpolynucleotides encoding the BDD FVIII protein described herein. In oneembodiment, the optimized coding sequences for the BDD FVIII protein isoperably linked to an expression control sequence. As used herein, twonucleic acid sequences are operably linked when they are covalentlylinked in such a way as to permit each component nucleic acid sequenceto retain its functionality. A coding sequence and a gene expressioncontrol sequence are said to be operably linked when they are covalentlylinked in such a way as to place the expression or transcription and/ortranslation of the coding sequence under the influence or control of thegene expression control sequence. Two DNA sequences are said to beoperably linked if induction of a promoter in the 5′ gene expressionsequence results in the transcription of the coding sequence and if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region to direct the transcription of the codingsequence, or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a gene expressionsequence would be operably linked to a coding nucleic acid sequence ifthe gene expression sequence were capable of effecting transcription ofthat coding nucleic acid sequence such that the resulting transcript istranslated into the desired protein or polypeptide.

Viral vectors include, but are not limited to, nucleic acid sequencesfrom the following viruses: retrovirus, such as Moloney murine leukemiavirus, Harvey murine sarcoma virus, murine mammary tumor virus, and Roussarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses;polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus;vaccinia virus; polio virus; and RNA virus such as a retrovirus. One canreadily employ other vectors well-known in the art. Certain viralvectors are based on non-cytopathic eukaryotic viruses in whichnon-essential genes have been replaced with the gene of interest.Non-cytopathic viruses include retroviruses, the life cycle of whichinvolves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman Co., New York (1990) and Murry, E. J., Methods in MolecularBiology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, adouble-stranded DNA virus. The adeno-associated virus can be engineeredto be replication-deficient and is capable of infecting a wide range ofcell types and species. It further has advantages such as heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hematopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operably encoded within the plasmid. Somecommonly used plasmids available from commercial suppliers includepBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMVplasmids, pSV40, and pBlueScript. Additional examples of specificplasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro,catalog number V87020; pcDNA4/myc-His, catalog number V86320; andpBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad,Calif.). Other plasmids are well-known to those of ordinary skill in theart. Additionally, plasmids can be custom designed using standardmolecular biology techniques to remove and/or add specific fragments ofDNA.

Host Cells

The invention also provides host cells comprising the isolated nucleicacid molecules of the invention. As used herein, the term“transformation” shall be used in a broad sense to refer to theintroduction of DNA into a recipient host cell that changes the genotypeand consequently results in a change in the recipient cell.

“Host cells” refers to cells that have been transformed with vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. The host cells of the present invention arepreferably of mammalian origin; most preferably of human or mouseorigin. Those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for their purpose. Exemplary host cell lines include, but are notlimited to, CHO, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFRminus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS(a derivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT(bovine endothelial cells), RAJI (human lymphocyte), PER.C6®, NS0, CAP,BHK21, and HEK 293 (human kidney). Host cell lines are typicallyavailable from commercial services, the American Tissue CultureCollection, or from published literature.

Introduction of the isolated nucleic acid molecules of the inventioninto the host cell can be accomplished by various techniques well knownto those of skill in the art, These include, but are not limited to,transfection (including electrophoresis and electroporation), protoplastfusion, calcium phosphate precipitation, cell fusion with enveloped DNA,microinjection, and infection with intact virus. See, Ridgway, A. A. G.“Mammalian Expression Vectors” Chapter 24.2, pp. 470-472 Vectors,Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Mostpreferably, plasmid introduction into the host is via electroporation.The transformed cells are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

Host cells comprising the isolated nucleic acid molecules of theinvention are grown in an appropriate growth medium. As used herein, theterm “appropriate growth medium” means a medium containing nutrientsrequired for the growth of cells. Nutrients required for cell growth caninclude a carbon source, a nitrogen source, essential amino acids,vitamins, minerals, and growth factors. Optionally, the media cancontain one or more selection factors. Optionally the media can containbovine calf serum or fetal calf serum (FCS). In one embodiment, themedia contains substantially no IgG. The growth medium will generallyselect for cells containing the DNA construct by, for example, drugselection or deficiency in an essential nutrient which is complementedby the selectable marker on the DNA construct or co-transfected with theDNA construct. Cultured mammalian cells are generally grown incommercially available serum-containing or serum-free media (e.g., MEM,DMEM, DMEM/F12). In one embodiment, the medium is CDoptiCHO (Invitrogen,Carlsbad, Calif.). In another embodiment, the medium is CD17(Invitrogen, Carlsbad, Calif.). Selection of a medium appropriate forthe particular cell line used is within the level of those ordinaryskilled in the art.

Preparation of Polypeptides

The invention also provides a polypeptide encoded by the isolatednucleic acid molecules of the invention. In other embodiments, thepolypeptide of the invention is encoded by a vector comprising theisolated nucleic molecules of the invention. In yet other embodiments,the polypeptide of the invention is produced by a host cell comprisingthe isolated nucleic molecules of the invention.

In other embodiments, the invention also provides a method of producinga polypeptide with FVIII activity, comprising culturing a host cell ofthe invention under conditions whereby a polypeptide with FVIII activityis produced, and recovering the polypeptide with FVIII activity. In someembodiments, the expression of the polypeptide with FVIII activity isincreased relative to a host cell cultured under the same conditions butcontaining a reference nucleotide sequence comprising SEQ ID NO:3, theparental FVIII gene sequence.

In other embodiments, the invention provides a method of increasing theexpression of a polypeptide with FVIII activity comprising culturing ahost cell of the invention under conditions whereby a polypeptide withFVIII activity is expressed by the nucleic acid molecule, wherein theexpression of the polypeptide with FVIII activity is increased relativeto a host cell cultured under the same conditions comprising a referencenucleic acid molecule comprising SEQ ID NO:3.

In other embodiments, the invention provides a method of improving yieldof a polypeptide with Factor VIII activity comprising culturing a hostcell under conditions whereby a polypeptide with Factor VIII activity isproduced by the nucleic acid molecule, wherein the yield of polypeptidewith Factor VIII activity is increased relative to a host cell culturedunder the same conditions comprising a reference nucleic acid sequencecomprising SEQ ID NO: 3.

A variety of methods are available for recombinantly producing a FVIIIprotein from the optimized nucleic acid molecule of the invention. Apolynucleotide of the desired sequence can be produced by de novosolid-phase DNA synthesis or by PCR mutagenesis of an earlier preparedpolynucleotide. Oligonucleotide-mediated mutagenesis is one method forpreparing a substitution, insertion, deletion, or alteration (e.g.,altered codon) in a nucleotide sequence. For example, the starting DNAis altered by hybridizing an oligonucleotide encoding the desiredmutation to a single-stranded DNA template. After hybridization, a DNApolymerase is used to synthesize an entire second complementary strandof the template that incorporates the oligonucleotide primer. In oneembodiment, genetic engineering, e.g., primer-based PCR mutagenesis, issufficient to incorporate an alteration, as defined herein, forproducing a polynucleotide of the invention.

For recombinant protein production, an optimized polynucleotide sequenceof the invention encoding the FVIII protein is inserted into anappropriate expression vehicle, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence, or in the case of an RNA viral vector, the necessaryelements for replication and translation.

The polynucleotide sequence of the invention is inserted into the vectorin proper reading frame. The expression vector is then transfected intoa suitable target cell which will express the polypeptide. Transfectiontechniques known in the art include, but are not limited to, calciumphosphate precipitation (Wigler et al. 1978, Cell 14: 725) andelectroporation (Neumann et al. 1982, EMBO, J 1: 841). A variety ofhost-expression vector systems can be utilized to express the FVIIIproteins described herein in eukaryotic cells. In one embodiment, theeukaryotic cell is an animal cell, including mammalian cells (e.g.HEK293 cells, PER.C6®, CHO, BHK, Cos, HeLa cells). A polynucleotidesequence of the invention can also code for a signal sequence that willpermit the FVIII protein to be secreted. One skilled in the art willunderstand that while the FVIII protein is translated the signalsequence is cleaved by the cell to form the mature protein. Varioussignal sequences are known in the art, e.g., native factor VII signalsequence, native factor IX signal sequence and the mouse IgK light chainsignal sequence. Alternatively, where a signal sequence is not includedthe FVIII protein can be recovered by lysing the cells.

The FVIII protein of the invention can be synthesized in a transgenicanimal, such as a rodent, goat, sheep, pig, or cow. The term “transgenicanimals” refers to non-human animals that have incorporated a foreigngene into their genome. Because this gene is present in germlinetissues, it is passed from parent to offspring. Exogenous genes areintroduced into single-celled embryos (Brinster et al. 1985, Proc. Natl.Acad. Sci. USA 82:4438). Methods of producing transgenic animals areknown in the art including transgenics that produce immunoglobulinmolecules (Wagner et al. 1981, Proc. Natl. Acad. Sci. USA 78: 6376;McKnight et al. 1983, Cell 34: 335; Brinster et al. 1983, Nature 306:332; Ritchie et al. 1984, Nature 312: 517; Baldassarre et al. 2003,Theriogenology 59: 831; Robl et al. 2003, Theriogenology 59: 107;Malassagne et al. 2003, Xenotransplantation 10 (3): 267).

The expression vectors can encode for tags that permit for easypurification or identification of the recombinantly produced protein.Examples include, but are not limited to, vector pUR278 (Ruther et al.1983, EMBO J. 2: 1791) in which the FVIII protein described hereincoding sequence can be ligated into the vector in frame with the lac Zcoding region so that a hybrid protein is produced; pGEX vectors can beused to express proteins with a glutathione S-transferase (GST) tag.These proteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The vectors include cleavage sites (e.g.,PreCission Protease (Pharmacia, Peapack, N.J.)) for easy removal of thetag after purification.

For the purposes of this invention, numerous expression vector systemscan be employed. These expression vectors are typically replicable inthe host organisms either as episomes or as an integral part of the hostchromosomal DNA. Expression vectors can include expression controlsequences including, but not limited to, promoters (e.g.,naturally-associated or heterologous promoters), enhancers, signalsequences, splice signals, enhancer elements, and transcriptiontermination sequences. Preferably, the expression control sequences areeukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells. Expression vectors can also utilizeDNA elements which are derived from animal viruses such as bovinepapilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus,retroviruses (RSV, MMTV or MOMLV), cytomegalovirus (CMV), or SV40 virus.Others involve the use of polycistronic systems with internal ribosomebinding sites.

Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362). Cells which have integrated the DNA into their chromosomescan be selected by introducing one or more markers which allow selectionof transfected host cells. The marker can provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation.

An example of a vector useful for expressing an optimized FVIII sequenceis NEOSPLA (U.S. Pat. No. 6,159,730). This vector contains thecytomegalovirus promoter/enhancer, the mouse beta globin major promoter,the SV40 origin of replication, the bovine growth hormonepolyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2,the dihydrofolate reductase gene and leader sequence. This vector hasbeen found to result in very high level expression of antibodies uponincorporation of variable and constant region genes, transfection incells, followed by selection in G418 containing medium and methotrexateamplification. Vector systems are also taught in U.S. Pat. Nos.5,736,137 and 5,658,570, each of which is incorporated by reference inits entirety herein. This system provides for high expression levels,e.g., >30 pg/cell/day. Other exemplary vector systems are disclosede.g., in U.S. Pat. No. 6,413,777.

In other embodiments the polypeptides of the invention of the instantinvention can be expressed using polycistronic constructs. In theseexpression systems, multiple gene products of interest such as multiplepolypeptides of multimer binding protein can be produced from a singlepolycistronic construct. These systems advantageously use an internalribosome entry site (IRES) to provide relatively high levels ofpolypeptides in eukaryotic host cells. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein.

More generally, once the vector or DNA sequence encoding a polypeptidehas been prepared, the expression vector can be introduced into anappropriate host cell. That is, the host cells can be transformed.Introduction of the plasmid into the host cell can be accomplished byvarious techniques well known to those of skill in the art, as discussedabove. The transformed cells are grown under conditions appropriate tothe production of the FVIII polypeptide, and assayed for FVIIIpolypeptide synthesis. Exemplary assay techniques include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

In descriptions of processes for isolation of polypeptides fromrecombinant hosts, the terms “cell” and “cell culture” are usedinterchangeably to denote the source of polypeptide unless it is clearlyspecified otherwise. In other words, recovery of polypeptide from the“cells” can mean either from spun down whole cells, or from the cellculture containing both the medium and the suspended cells.

The host cell line used for protein expression is preferably ofmammalian origin; most preferably of human or mouse origin, as theisolated nucleic acids of the invention have been optimized forexpression in human cells. Exemplary host cell lines have been describedabove. In one embodiment of the method to produce a polypeptide withFVIII activity, the host cell is a HEK293 cell. In another embodiment ofthe method to produce a polypeptide with FVIII activity, the host cellis a CHO cell.

Genes encoding the polypeptides of the invention can also be expressedin non-mammalian cells such as bacteria or yeast or plant cells. In thisregard it will be appreciated that various unicellular non-mammalianmicroorganisms such as bacteria can also be transformed; i.e., thosecapable of being grown in cultures or fermentation. Bacteria, which aresusceptible to transformation, include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the polypeptides typically become part ofinclusion bodies. The polypeptides must be isolated, purified and thenassembled into functional molecules.

Alternatively, optimized nucleotide sequences of the invention can beincorporated in transgenes for introduction into the genome of atransgenic animal and subsequent expression in the milk of thetransgenic animal (see, e.g., Deboer et al., U.S. Pat. No. 5,741,957,Rosen, U.S. Pat. No. 5,304,489, and Meade el al., U.S. Pat. No.5,849,992). Suitable transgenes include coding sequences forpolypeptides in operable linkage with a promoter and enhancer from amammary gland specific gene, such as casein or beta lactoglobulin.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.An affinity tag sequence (e.g. a His(6) tag) can optionally be attachedor included within the polypeptide sequence to facilitate downstreampurification.

Once expressed, the FVIII protein can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity column chromatography, HPLC purification, gel electrophoresisand the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure proteins of at leastabout 90 to 95% homogeneity are preferred, and 98 to 99% or morehomogeneity most preferred, for pharmaceutical uses.

Pharmaceutical Composition

Compositions containing the FVIII protein of the present invention orthe isolated nucleic acids of the present invention can contain asuitable pharmaceutically acceptable carrier. For example, they cancontain excipients and/or auxiliaries that facilitate processing of theactive compounds into preparations designed for delivery to the site ofaction.

The pharmaceutical composition can be formulated for parenteraladministration (i.e. intravenous, subcutaneous, or intramuscular) bybolus injection. Formulations for injection can be presented in unitdosage form, e.g., in ampoules or in multidose containers with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, e.g., pyrogen free water.

Suitable formulations for parenteral administration also include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions can be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions can contain substances,which increase the viscosity of the suspension, including, for example,sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, thesuspension can also contain stabilizers. Liposomes also can be used toencapsulate the molecules of the invention for delivery into cells orinterstitial spaces. Exemplary pharmaceutically acceptable carriers arephysiologically compatible solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like. In some embodiments, the composition comprisesisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride. In other embodiments, the compositionscomprise pharmaceutically acceptable substances such as wetting agentsor minor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the active ingredients.

Compositions of the invention can be in a variety of forms, including,for example, liquid (e.g., injectable and infusible solutions),dispersions, suspensions, semi-solid and solid dosage forms. Thepreferred form depends on the mode of administration and therapeuticapplication.

The composition can be formulated as a solution, micro emulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the active ingredient in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active ingredient into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The active ingredient can be formulated with a controlled-releaseformulation or device. Examples of such formulations and devices includeimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, for example, ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for the preparation ofsuch formulations and devices are known in the art. See e.g., Sustainedand Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,Marcel Dekker, Inc., New York, 1978.

Injectable depot formulations can be made by forming microencapsulatedmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the polymer employed, the rate of drug release can becontrolled. Other exemplary biodegradable polymers are polyorthoestersand polyanhydrides. Depot injectable formulations also can be preparedby entrapping the drug in liposomes or microemulsions.

Supplementary active compounds can be incorporated into thecompositions. In one embodiment, the chimeric protein of the inventionis formulated with another clotting factor, or a variant, fragment,analogue, or derivative thereof. For example, the clotting factorincludes, but is not limited to, factor V, factor VII, factor VIII,factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin,fibrinogen, von Willebrand factor or recombinant soluble tissue factor(rsTF) or activated forms of any of the preceding. The clotting factorof hemostatic agent can also include anti-fibrinolytic drugs, e.g.,epsilon-amino-caproic acid, tranexamic acid.

Dosage regimens can be adjusted to provide the optimum desired response.For example, a single bolus can be administered, several divided dosescan be administered over time, or the dose can be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton,Pa. 1980).

In addition to the active compound, the liquid dosage form can containinert ingredients such as water, ethyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan.

Non-limiting examples of suitable pharmaceutical carriers are alsodescribed in Remington's Pharmaceutical Sciences by E. W. Martin. Someexamples of excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol, and the like. The composition canalso contain pH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations can also include flavoring, coloring andsweetening agents. Alternatively, the composition can be presented as adry product for constitution with water or another suitable vehicle.

For buccal administration, the composition can take the form of tabletsor lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of a nebulizedaerosol with or without excipients or in the form of an aerosol sprayfrom a pressurized pack or nebulizer, with optionally a propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator can be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In one embodiment, a pharmaceutical composition comprises a FVIIIprotein, the optimized polynucleotide encoding the FVIII protein, thevector comprising the polynucleotide, or the host cell comprising thevector, and a pharmaceutically acceptable carrier. In some embodiments,the composition is administered by a route selected from the groupconsisting of topical administration, intraocular administration,parenteral administration, intrathecal administration, subduraladministration and oral administration. The parenteral administrationcan be intravenous or subcutaneous administration.

In other embodiments, the composition is used to treat a bleedingdisease or condition in a subject in need thereof. The bleeding diseaseor condition is selected from the group consisting of a bleedingcoagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, bleeding in the illiopsoas sheathand any combinations thereof. In still other embodiments, the subject isscheduled to undergo a surgery. In yet other embodiments, the treatmentis prophylactic or on-demand.

Methods of Treatment

The invention provides a method of treating a bleeding disordercomprising administering to a subject in need thereof a nucleic acidmolecule, vector, or polypeptide of the invention. In some embodiments,the bleeding disorder is characterized by a deficiency in Factor VIII.In some embodiments, the bleeding disorder is hemophilia. In someembodiments, the bleeding disorder is hemophilia A. In some embodimentsof the method of treating a bleeding disorder, plasma Factor VIIIactivity at 24 hours post administration is increased relative to asubject administered a reference nucleic acid molecule comprising SEQ IDNO: 3, a vector comprising the reference nucleic acid molecule, or apolypeptide encoded by the reference nucleic acid molecule.

The invention also relates to a method of treating, ameliorating, orpreventing a hemostatic disorder in a subject comprising administering atherapeutically effective amount of a FVIII protein of the invention oran isolated nucleic acid molecule of the invention. The treatment,amelioration, and prevention by the FVIII protein or isolated nucleicacid molecule can be a bypass therapy. The subject receiving bypasstherapy can have already developed an inhibitor to a clotting factor,e.g., Factor VIII, or is subject to developing a clotting factorinhibitor.

The nucleic acid molecules, vectors, or FVIII polypeptides of theinvention treat or prevent a hemostatic disorder by promoting theformation of a fibrin clot. The FVIII protein of the invention canactivate a member of a coagulation cascade. The clotting factor can be aparticipant in the extrinsic pathway, the intrinsic pathway or both.

The nucleic acid molecules, vectors, or FVIII polypeptides of theinvention can be used to treat hemostatic disorders known to betreatable with FVIII. The hemostatic disorders that can be treated usingmethods of the invention include, but are not limited to, hemophilia A,hemophilia B, von Willebrand's disease, Factor XI deficiency (PTAdeficiency), Factor XII deficiency, as well as deficiencies orstructural abnormalities in fibrinogen, prothrombin, Factor V, FactorVII, Factor X, or Factor XIII, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath. Compositions for administration to a subject include nucleicacid molecules which comprise an optimized nucleotide sequence of theinvention encoding a FVIII clotting factor (for gene therapyapplications) as well as FVIII polypeptide molecules.

In some embodiments, the hemostatic disorder is an inherited disorder.In one embodiment, the subject has hemophilia A. In other embodiments,the hemostatic disorder is the result of a deficiency in Factor VIII. Inother embodiments, the hemostatic disorder can be the result of adefective FVIII clotting factor.

In another embodiment, the hemostatic disorder can be an acquireddisorder. The acquired disorder can result from an underlying secondarydisease or condition. The unrelated condition can be, as an example, butnot as a limitation, cancer, an autoimmune disease, or pregnancy. Theacquired disorder can result from old age or from medication to treat anunderlying secondary disorder (e.g. cancer chemotherapy).

The invention also relates to methods of treating a subject that doesnot have a hemostatic disorder or a secondary disease or conditionresulting in acquisition of a hemostatic disorder. The invention thusrelates to a method of treating a subject in need of a generalhemostatic agent comprising administering a therapeutically effectiveamount of the FVIII polypeptide of the invention or an isolated nucleicacid molecule of the invention. For example, in one embodiment, thesubject in need of a general hemostatic agent is undergoing, or is aboutto undergo, surgery. The FVIII polypeptide of the invention or anisolated nucleic acid molecule of the invention can be administeredprior to or after surgery as a prophylactic. The FVIII polypeptide ofthe invention or an isolated nucleic acid molecule of the invention canbe administered during or after surgery to control an acute bleedingepisode. The surgery can include, but is not limited to, livertransplantation, liver resection, or stem cell transplantation.

In another embodiment, the FVIII polypeptide of the invention or anisolated nucleic acid molecule of the invention can be used to treat asubject having an acute bleeding episode who does not have a hemostaticdisorder. The acute bleeding episode can result from severe trauma,e.g., surgery, an automobile accident, wound, laceration gun shot, orany other traumatic event resulting in uncontrolled bleeding.

The FVIII protein or the isolated nucleic acid molecules of theinvention can be used to prophylactically treat a subject with ahemostatic disorder. The FVIII protein or the isolated nucleic acidmolecules of the invention can be used to treat an acute bleedingepisode in a subject with a hemostatic disorder.

In some embodiments, a FVIII protein composition of the invention isadministered in combination with at least one other agent that promoteshemostasis. Said other agent that promotes hemostasis in a therapeuticwith demonstrated clotting activity. As an example, but not as alimitation, the hemostatic agent can include Factor V, Factor VII,Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, prothrombin, orfibrinogen or activated forms of any of the preceding. The clottingfactor or hemostatic agent can also include anti-fibrinolytic drugs,e.g., epsilon-amino-caproic acid, tranexamic acid.

In one embodiment of the invention, the composition (e.g., the FVIIIpolypeptide or the optimized nucleic acid molecule encoding the FVIIIpolypeptide) is one in which the FVIII is present in activatable formwhen administered to a subject. Such an activatable molecule can beactivated in vivo at the site of clotting after administration to asubject.

The FVIII polypeptide or the optimized nucleic acid molecule encodingthe FVIII polypeptide can be administered intravenously, subcutaneously,intramuscularly, or via any mucosal surface, e.g., orally, sublingually,buccally, sublingually, nasally, rectally, vaginally or via pulmonaryroute. The FVIII protein can be implanted within or linked to abiopolymer solid support that allows for the slow release of thechimeric protein to the desired site.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g. sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g. almond oil, oilyesters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid).The preparations can also include flavoring, coloring and sweeteningagents. Alternatively, the composition can be presented as a dry productfor constitution with water or another suitable vehicle.

For buccal and sublingual administration the composition can take theform of tablets, lozenges or fast dissolving films according toconventional protocols.

For administration by inhalation, the polypeptide having FVIII activityfor use according to the present invention are conveniently delivered inthe form of an aerosol spray from a pressurized pack or nebulizer (e.g.in PBS), with a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

In one embodiment, the route of administration of the FVIII polypeptideor the optimized nucleic acid molecule encoding the FVIII polypeptide isparenteral. The term parenteral as used herein includes intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal orvaginal administration. The intravenous form of parenteraladministration is preferred. While all these forms of administration areclearly contemplated as being within the scope of the invention, a formfor administration would be a solution for injection, in particular forintravenous or intraarterial injection or drip. Usually, a suitablepharmaceutical composition for injection can comprise a buffer (e.g.acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate),optionally a stabilizer agent (e.g. human albumin), etc. However, inother methods compatible with the teachings herein, the FVIIIpolypeptides or the optimized nucleic acid molecules encoding the FVIIIpolypeptides can be delivered directly to the site of the adversecellular population thereby increasing the exposure of the diseasedtissue to the therapeutic agent.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives can also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., a polypeptide by itself or incombination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations can be packaged and sold in the form of a kit. Sucharticles of manufacture will preferably have labels or package insertsindicating that the associated compositions are useful for treating asubject suffering from, or predisposed to clotting disorders.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

Effective doses of the compositions of the present invention, for thetreatment of conditions vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human but non-human mammalsincluding transgenic mammals can also be treated. Treatment dosages canbe titrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

Dosages can range from 1000 ug/kg to 0.1 ng/kg body weight. In oneembodiment, the dosing range is 1 ug/kg to 100 ug/kg. The FVIIIpolypeptide or the optimized nucleic acid molecule encoding the FVIIIpolypeptide can be administered continuously or at specific timedintervals. In vitro assays can be employed to determine optimal doseranges and/or schedules for administration. In vitro assays that measureclotting factor activity are known in the art. Additionally, effectivedoses can be extrapolated from dose-response curves obtained from animalmodels, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99 (8); 2670).

Doses intermediate in the above ranges are also intended to be withinthe scope of the invention. Subjects can be administered such dosesdaily, on alternative days, weekly or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. In some methods, two or more polypeptides can beadministered simultaneously, in which case the dosage of eachpolypeptide administered falls within the ranges indicated.

FVIII polypeptides or the optimized nucleic acid molecules encoding theFVIII polypeptides of the invention can be administered on multipleoccasions. Intervals between single dosages can be daily, weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of modified polypeptide or antigen in thepatient. Alternatively, polypeptides can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of thepolypeptide or polynucleotide in the patient.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the FVIII polypeptide or theoptimized nucleic acid molecule encoding the FVIII polypeptide or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance or minimize effects ofdisease. Such an amount is defined to be a “prophylactic effectivedose.” A relatively low dosage is administered at relatively infrequentintervals over a long period of time. Some patients continue to receivetreatment for the rest of their lives.

FVIII polypeptides or the optimized nucleic acid molecules encoding theFVIII polypeptides of the invention can optionally be administered incombination with other agents that are effective in treating thedisorder or condition in need of treatment (e.g., prophylactic ortherapeutic).

As used herein, the administration of FVIII polypeptides or theoptimized nucleic acid molecules encoding the FVIII polypeptides of theinvention in conjunction or combination with an adjunct therapy meansthe sequential, simultaneous, coextensive, concurrent, concomitant orcontemporaneous administration or application of the therapy and thedisclosed polypeptides. Those skilled in the art will appreciate thatthe administration or application of the various components of thecombined therapeutic regimen can be timed to enhance the overalleffectiveness of the treatment. A skilled artisan (e.g. a physician)would be readily be able to discern effective combined therapeuticregimens without undue experimentation based on the selected adjuncttherapy and the teachings of the instant specification.

It will further be appreciated that the FVIII polypeptide or theoptimized nucleic acid molecule encoding the FVIII polypeptide of theinstant invention can be used in conjunction or combination with anagent or agents (e.g. to provide a combined therapeutic regimen).Exemplary agents with which a polypeptide or polynucleotide of theinvention can be combined include agents that represent the currentstandard of care for a particular disorder being treated. Such agentscan be chemical or biologic in nature. The term “biologic” or “biologicagent” refers to any pharmaceutically active agent made from livingorganisms and/or their products which is intended for use as atherapeutic.

The amount of agent to be used in combination with the polynucleotidesor polypeptides of the instant invention can vary by subject or can beadministered according to what is known in the art. See for example,Bruce A Chabner et al., Antineoplastic Agents, in GOODMAN & GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Hardman etal., eds., 9^(th) ed. 1996). In another embodiment, an amount of such anagent consistent with the standard of care is administered.

As previously discussed, the polynucleotides and polypeptides of thepresent invention, can be administered in a pharmaceutically effectiveamount for the in vivo treatment of clotting disorders. In this regard,it will be appreciated that the polypeptides or polynucleotides of theinvention can be formulated to facilitate administration and promotestability of the active agent. Preferably, pharmaceutical compositionsin accordance with the present invention comprise a pharmaceuticallyacceptable, non-toxic, sterile carrier such as physiological saline,non-toxic buffers, preservatives and the like. Of course, thepharmaceutical compositions of the present invention can be administeredin single or multiple doses to provide for a pharmaceutically effectiveamount of the polypeptide.

A number of tests are available to assess the function of thecoagulation system: activated partial thromboplastin time (aPTT) test,chromogenic assay, ROTEM® assay, prothrombin time (PT) test (also usedto determine INR), fibrinogen testing (often by the Clauss method),platelet count, platelet function testing (often by PFA-100), TCT,bleeding time, mixing test (whether an abnormality corrects if thepatient's plasma is mixed with normal plasma), coagulation factorassays, antiphosholipid antibodies, D-dimer, genetic tests (e.g., factorV Leiden, prothrombin mutation G20210A), dilute Russell's viper venomtime (dRVVT), miscellaneous platelet function tests, thromboelastography(TEG or Sonoclot), thromboelastometry (TEM®, e.g, ROTEM®), or euglobulinlysis time (ELT).

The aPTT test is a performance indicator measuring the efficacy of boththe “intrinsic” (also referred to the contact activation pathway) andthe common coagulation pathways. This test is commonly used to measureclotting activity of commercially available recombinant clottingfactors, e.g., FVIII or FIX. It is used in conjunction with prothrombintime (PT), which measures the extrinsic pathway.

ROTEM® analysis provides information on the whole kinetics ofhaemostasis: clotting time, clot formation, clot stability and lysis.The different parameters in thromboelastometry are dependent on theactivity of the plasmatic coagulation system, platelet function,fibrinolysis, or many factors which influence these interactions. Thisassay can provide a complete view of secondary haemostasis.

Gene Therapy

The invention provides a method of increasing expression of apolypeptide with Factor VIII activity in a subject comprisingadministering the isolated nucleic acid molecule of the invention to asubject in need thereof, wherein the expression of the polypeptide isincreased relative to a reference nucleic acid molecule comprising SEQID NO: 3. The invention also provides a method of increasing expressionof a polypeptide with Factor VIII activity in a subject comprisingadministering a vector of the invention to a subject in need thereof,wherein the expression of the polypeptide is increased relative to avector comprising a reference nucleic acid molecule.

Somatic gene therapy has been explored as a possible treatment forhemophilia A. Gene therapy is a particularly appealing treatment forhemophilia because of its potential to cure the disease throughcontinuous endogenous production of FVIII following a singleadministration of vector. Haemophilia A is well suited for a genereplacement approach because its clinical manifestations are entirelyattributable to the lack of a single gene product (FVIII) thatcirculates in minute amounts (200 ng/ml) in the plasma.

A FVIII protein of the invention can be produced in vivo in a mammal,e.g., a human patient, using a gene therapy approach to treatment of ableeding disease or disorder selected from the group consisting of ableeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed,hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, traumacapitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath would be therapeutically beneficial. In one embodiment, thebleeding disease or disorder is hemophilia. In another embodiment, thebleeding disease or disorder is hemophilia A. This involvesadministration of an optimized FVIII encoding nucleic acid operablylinked to suitable expression control sequences. In certain embodiment,these sequences are incorporated into a viral vector. Suitable viralvectors for such gene therapy include adenoviral vectors, lentiviralvectors, baculoviral vectors, Epstein Barr viral vectors, papovaviralvectors, vaccinia viral vectors, herpes simplex viral vectors, and adenoassociated virus (AAV) vectors. The viral vector can be areplication-defective viral vector. In other embodiments, an adenoviralvector has a deletion in its E1 gene or E3 gene. When an adenoviralvector is used, the mammal can not be exposed to a nucleic acid encodinga selectable marker gene. In other embodiments, the sequences areincorporated into a non-viral vector known to those skilled in the art.

All of the various aspects, embodiments, and options described hereincan be combined in any and all variations.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

Having generally described this invention, a further understanding canbe obtained by reference to the examples provided herein. These examplesare for purposes of illustration only and are not intended to belimiting.

EXAMPLES

Two codon-optimized BDD FVIII sequences were designed with the followinggoals:

1. Remove all matrix attachment-like region (MAR) sequences (ATATTT andAAATAT; SEQ ID NOs: 5 and 6, respectively);2. Remove all destabilizing sequences (ATTTA, SEQ ID NO:8 and TAAAT, SEQID NO:9);3. Remove promoter binding sequences (TATAA, SEQ ID NO:12 and TTATA, SEQID NO:13);4. Remove AU-rich sequence elements (AREs): ATTTTATT (nucleotide 2468)and ATTTTTAA (nucleotide 3790) (SEQ ID NOs: 14 and 15, respectively);5. Add kozak sequence (GCCGCCACCATGC, underlined indicate thetranslation start codon; SEQ ID NO: 16) to increase translationalinitiation;6. Adjust restriction sites to facilitate cloning;7. Adapt codon usage to the codon bias of Homo sapiens genes;8. Adjust to avoid regions of very high (>70%) or low (<30%) GC content,which can increase RNA stability or prolong RNA half-life.

Example 1: Codon Optimization by GENSCRIPT OPTIMUMGENE™

The BDD FVIII nucleotide sequence was codon optimized using GENSCRIPTOPTIMUMGENE™ codon optimization technology. (GenScript Corp., NewJersey, USA). The GENSCRIPT OPTIMUMGENE™ codon optimization technologyis described in Burgess-Brown et al., Protein Expr Purif. 59(1):94-102(2008).

The following human codon usage data was used for optimization:

CODON AMINO ACID FRACTION FREQUENCY/THOUSAND TTT F 0.45 16.9 TCT S 0.1814.6 TAT Y 0.43 12.0 TGT C 0.45 9.9 TTC F 0.55 20.4 TCC S 0.22 17.4 TACY 0.57 15.6 TGC C 0.55 12.2 TTA L 0.07 7.2 TCA S 0.15 11.7 TAA * 0.280.7 TGA * 0.52 1.3 TTG L 0.13 12.6 TCG S 0.06 4.5 TAG * 0.20 0.5 TGG W1.00 12.8 CTT L 0.13 12.8 CCT P 0.28 17.3 CAT H 0.41 10.4 CGT R 0.08 4.7CTC L 0.20 19.4 CCC P 0.33 20.0 CAC H 0.59 14.9 CGC R 0.19 10.9 CTA L0.07 6.9 CCA P 0.27 16.7 CAA Q 0.25 11.8 CGA R 0.11 6.3 CTG L 0.41 40.3CCG P 0.11 7.0 CAG Q 0.75 34.6 CGG R 0.21 11.9 ATT I 0.36 15.7 ACT T0.24 12.8 AAT N 0.46 16.7 AGT S 0.15 11.9 ATC I 0.48 21.4 ACC T 0.3619.2 AAC N 0.54 19.5 AGC S 0.24 19.4 ATA I 0.16 7.1 ACA T 0.28 14.8 AAAK 0.42 24.0 AGA R 0.20 11.5 ATG M 1.00 22.3 ACG T 0.12 6.2 AAG K 0.5832.9 AGG R 0.20 11.4 GTT V 0.18 10.9 GCT A 0.26 18.6 GAT D 0.46 22.3 GGTG 0.16 10.8 GTC V 0.24 14.6 GCC A 0.40 28.5 GAC D 0.54 26.0 GGC G 0.3422.8 GTA V 0.11 7.0 GCA A 0.23 16.0 GAA E 0.42 29.0 GGA G 0.25 16.3 GTGV 0.47 28.9 GCG A 0.11 7.6 GAG E 0.58 40.8 GGG G 0.25 16.4

Codon usage was adjusted to human bias with the human codon adaptationindex (CAI) changing from 0.75 (wild type BDD FVIII) to 0.88 (GenScriptoptimized BDD FVIII). G/C content was increased from 46.16% to 51.56%.Peaks of G/C content in a 60 bp window were removed. The resultingsequence of GenScript optimized BDD FVIII is disclosed herein as SEQ IDNO:1 and is shown in FIG. 2.

Example 2: Codon Optimization by GENEART® GENEOPTIMIZER®

The BDD FVIII nucleotide sequence was codon optimized using GENEART®GENEOPTIMIZER® software. (Invitrogen LIFE TECHNOLOGIES™ Corp., GrandIsland, N.Y.). The GENEART® GENEOPTIMIZER® codon optimization technologyis described in Graf et al., J. Virol. 74(22):10822-10826 (2000).

Codon usage was adjusted to human bias with the human codon adaptationindex (CAI) changing from 0.75 (wild type BDD FVIII) to 0.96 (GeneArtoptimized BDD FVIII). G/C content was increased from 46.16% to 59%. Theresulting sequence of GeneArt optimized BDD FVIII is disclosed herein asSEQ ID NO:2 and is shown in FIG. 3.

Example 3: Expression Constructs

All constructs were made in the Invitrogen pcDNA™4 vector backbone,which contains a human cytomegalovirus immediate-early (CMV) promoter, aQBI SP163 translation enhancer, and a ZEOCIN™ resistance gene forselection.

pSYN-FVIII-066 drives expression of wild-type BDD FVIII (SEQ ID NO:3) inpcDNA4 backbone (Invitrogen).

pSYN-FVIII-116 drives expression of codon-optimized BDD FVIII (SEQ IDNO:1) in pcDNA4 backbone. The construct is derived from pSYN-FVIII-066by replacing wild-type BDD FVIII with codon-optimized BDD FVIII (SEQ IDNO:1) using BsiWI and XhoI sites.

pSYN-FVIII-115 drives expression of codon-optimized BDD FVIII (SEQ IDNO:2) in pcDNA4 backbone. The construct is derived from pSYN-FVIII-066by replacing wild-type BDD FVIII with codon-optimized BDD FVIII (SEQ IDNO:2) using BsiWI and XhoI sites.

All constructs were confirmed by DNA sequencing.

Example 4: Codon Optimization Improves FVIII Expression in a HemA Mouse

To ask whether either codon optimized BDD FVIII constructs result inincreased FVIII protein expression, expression plasmids pSYN-FVIII116,pSYN-FVIII115, and wild type control pSYN-FVIII-066 were introduced intoHemA mice via hydrodynamic injection. Subsequently, FVIII expressionlevels were monitored in each injected mouse by plasma FVIII chromogenicassays.

Hydrodynamic injection is an efficient and safe non-viral method todeliver genes to the liver in small animals, such as mice and rats. Theprotein of interest is produced in the liver and can be detected within24 hours post-injection.

HemA mice weighing 20-35 grams were injected via intravenous tail veininjection with either pSYN-FVIII116, pSYN-FVIII115, or wild type controlpSYN-FVIII-066. Injections were made up with 10ug naked plasmid DNA freeof endotoxin in 0.9% sterile saline solution, to a total volume of 2 ml.Injections were performed rapidly, taking no more than 4-7 seconds toinject the full 2 ml DNA solution. Mice were closely monitored for twohours after injection, or until normal activity resumed. At 24 hourspost-injection, samples were collected via retro orbital bloodcollection, plasma was prepared and stored at −80° C. for furtheranalysis.

The FVIII activity was measured using the COATEST SP FVIII kit fromDiaPharma (lot # N089019) and all incubations were performed on a 37° C.plate heater with shaking. rFVIII standards ranged from 100 mIU/mL to0.78 mIU/mL. A pooled normal human plasma assay control and plasmasamples (diluted with 1× Coatest buffer) were added into Immulon 2HB96-well plates in duplicate (25 μL/well). Freshly preparedIXa/FX/Phospholipid mix (50 μL), 25 μL of 25 mM CaCl₂, and 50 μL of FXasubstrate were added sequentially into each well with a 5 minutesincubation between each addition. After incubating with the substrate,25 μL of 20% Acetic Acid was added to terminate the color reaction, andthe absorbance at OD405 was measured with a SpectraMAX plus (MolecularDevices) instrument. Data were analyzed with SoftMax Pro software(version 5.2). The Lowest Level of Quantification (LLOQ) is 7.8 mIU/mL.Results are shown in FIG. 7.

Low levels of BDD FVIII activity is detected upon administration ofFVIII expression plasmid pSYN-FVIII-066 (wild type BDD FVIII control).Average BDD FVIII activity in control mice is about 4-5 IU/mL (FIG. 7,circles). In contrast, on average about a three-fold increase in BDDFVIII activity is seen in the plasma of mice administered codonoptimized pSYN-FVIII115 or pSYN-FVIII116 (FIG. 7, squares andtriangles). Therefore, codon optimization of BDD FVIII by the approachesdescribed above improve FVIII expression in a HemA mouse model.

SEQUENCES optimized BDD FVIII SEQ ID NO: 1CGTACGGCCGCCACCATGCAGATTGAGCTGTCTACTTGCTTTTTCCTGTGCCTGCTGAGGTTTTGCTTTTCCGCTACACGAAGGTATTATCTGGGGGCTGTGGAACTGTCTTGGGATTACATGCAGAGTGACCTGGGAGAGCTGCCAGTGGACGCAAGGTTTCCCCCTAGAGTCCCTAAGTCATTCCCCTTCAACACTAGCGTGGTCTACAAGAAAACACTGTTCGTGGAGTTTACTGATCACCTGTTCAACATCGCAAAGCCTAGGCCACCCTGGATGGGACTGCTGGGGCCAACAATCCAGGCCGAGGTGTACGACACCGTGGTCATTACACTTAAGAACATGGCCTCACACCCCGTGAGCCTGCATGCTGTGGGCGTCAGCTACTGGAAGGCTTCCGAAGGAGCAGAGTATGACGATCAGACTTCCCAGAGAGAAAAAGAGGACGATAAGGTGTTTCCTGGCGGATCTCATACCTACGTGTGGCAGGTCCTGAAAGAGAATGGCCCTATGGCCTCCGACCCTCTGTGCCTGACCTACTCTTATCTGAGTCACGTGGACCTGGTCAAGGATCTGAACAGCGGCCTGATCGGAGCCCTGCTGGTGTGCAGGGAAGGAAGCCTGGCTAAGGAGAAAACCCAGACACTGCATAAGTTCATTCTGCTGTTCGCCGTGTTTGACGAAGGGAAATCATGGCACAGCGAGACAAAGAATAGTCTGATGCAGGACAGGGATGCCGCTTCAGCCAGAGCTTGGCCCAAAATGCACACTGTGAACGGCTACGTCAATCGCTCACTGCCTGGGCTGATCGGCTGCCACCGAAAGAGCGTGTATTGGCATGTCATCGGGATGGGCACCACACCTGAAGTGCACTCCATTTTCCTGGAGGGACATACCTTTCTGGTCCGCAACCACCGACAGGCTTCCCTGGAGATCTCTCCAATTACCTTCCTGACAGCACAGACTCTGCTGATGGACCTGGGGCAGTTCCTGCTGTTTTGCCACATCAGCTCCCACCAGCATGATGGCATGGAGGCTTACGTGAAAGTGGACTCTTGTCCCGAGGAACCTCAGCTGCGGATGAAGAACAATGAGGAAGCAGAAGACTATGACGATGACCTGACCGACTCCGAGATGGATGTGGTCCGATTCGATGACGATAACAGCCCCTCCTTTATCCAGATTAGATCTGTGGCCAAGAAACACCCTAAGACATGGGTCCATTACATCGCAGCCGAGGAAGAGGACTGGGATTATGCACCACTGGTGCTGGCACCAGACGATCGCTCCTACAAATCTCAGTATCTGAACAATGGGCCACAGAGGATTGGCAGAAAGTACAAGAAAGTGCGGTTCATGGCATATACCGATGAGACCTTCAAGACTCGCGAAGCCATCCAGCACGAGAGCGGCATCCTGGGACCACTGCTGTACGGAGAAGTGGGAGACACCCTGCTGATCATTTTCAAGAACCAGGCCAGCCGGCCTTACAATATCTATCCACATGGGATTACAGATGTGCGCCCTCTGTACAGCAGGAGACTGCCAAAGGGCGTCAAACACCTGAAGGACTTCCCAATCCTGCCCGGAGAAATCTTCAAGTACAAGTGGACTGTCACCGTCGAGGATGGCCCCACTAAGAGCGACCCTCGGTGCCTGACCCGCTACTATTCTAGTTTCGTGAATATGGAAAGAGATCTGGCAAGCGGACTGATCGGACCACTGCTGATTTGTTACAAAGAGAGCGTGGATCAGAGAGGCAACCAGATCATGTCCGACAAGCGGAATGTGATTCTGTTCAGTGTCTTTGACGAAAACAGGTCATGGTACCTGACCGAGAACATCCAGAGATTCCTGCCTAATCCAGCTGGGGTGCAGCTGGAAGATCCTGAGTTTCAGGCATCTAACATCATGCATAGTATTAATGGCTACGTGTTCGACAGTTTGCAGCTGAGCGTGTGCCTGCACGAGGTCGCTTACTGGTATATCCTGAGCATTGGGGCACAGACAGATTTCCTGAGCGTGTTCTTTTCCGGCTACACTTTTAAGCATAAAATGGTCTATGAGGACACACTGACTCTGTTCCCCTTCAGCGGCGAAACCGTGTTTATGAGCATGGAGAATCCCGGACTGTGGATTCTGGGGTGCCACAACAGCGATTTCAGAAATCGCGGAATGACTGCCCTGCTGAAAGTGTCAAGCTGTGACAAGAACACCGGGGACTACTATGAAGATTCATACGAGGACATCAGCGCATATCTGCTGTCCAAAAACAATGCCATTGAACCCCGGTCTTTTAGTCAGAATCCTCCAGTGCTGAAGCGGCACCAGCGCGAGATCACCCGCACTACCCTGCAGAGTGATCAGGAAGAGATCGACTACGACGATACAATTTCTGTGGAAATGAAGAAAGAGGACTTCGATATCTATGACGAAGATGAGAACCAGAGTCCTCGATCATTCCAGAAGAAAACCAGGCATTACTTTATTGCCGCAGTGGAGCGGCTGTGGGATTATGGCATGTCCTCTAGTCCTCACGTGCTGCGAAATAGGGCCCAGTCAGGAAGCGTCCCACAGTTCAAGAAAGTGGTCTTCCAGGAGTTTACAGACGGGTCCTTTACTCAGCCACTGTACAGGGGCGAACTGAACGAGCACCTGGGACTGCTGGGGCCCTATATCAGAGCAGAAGTGGAGGATAACATTATGGTCACCTTCAGAAATCAGGCCTCTCGGCCTTACAGTTTTTATTCAAGCCTGATCTCTTACGAAGAGGACCAGCGACAGGGAGCTGAACCACGAAAAAACTTCGTGAAGCCTAATGAGACCAAAACATACTTTTGGAAGGTGCAGCACCATATGGCCCCAACAAAAGACGAGTTCGATTGCAAGGCATGGGCCTATTTTTCTGACGTGGATCTGGAGAAGGACGTGCACAGTGGCCTGATTGGCCCACTGCTGGTGTGCCATACTAACACCCTGAATCCAGCCCACGGCCGGCAGGTCACTGTCCAGGAGTTCGCTCTGTTCTTTACCATCTTTGATGAGACAAAGAGCTGGTACTTCACCGAAAACATGGAGCGAAATTGCAGGGCTCCATGTAACATTCAGATGGAAGACCCCACATTCAAGGAGAACTACCGCTTTCATGCTATCAATGGATACATCATGGATACTCTGCCCGGGCTGGTCATGGCACAGGACCAGAGAATCCGGTGGTATCTGCTGAGCATGGGCAGCAACGAGAATATCCACTCAATTCATTTCAGCGGGCACGTGTTTACTGTCAGGAAGAAAGAAGAGTACAAGATGGCCCTGTACAACCTGTATCCCGGCGTGTTCGAAACCGTCGAGATGCTGCCTAGCAAGGCCGGAATCTGGAGAGTGGAATGCCTGATTGGAGAGCACCTGCATGCTGGGATGTCTACCCTGTTTCTGGTGTACAGTAATAAGTGTCAGACACCCCTGGGAATGGCATCCGGGCATATCAGGGATTTCCAGATTACCGCATCTGGACAGTACGGACAGTGGGCACCTAAGCTGGCTAGACTGCACTATTCCGGATCTATCAACGCTTGGTCCACAAAAGAGCCTTTCTCTTGGATTAAGGTGGACCTGCTGGCCCCAATGATCATTCATGGCATCAAAACTCAGGGAGCTCGGCAGAAGTTCTCCTCTCTGTACATCTCACAGTTTATCATCATGTACAGCCTGGATGGGAAGAAATGGCAGACATACCGCGGCAATAGCACAGGAACTCTGATGGTGTTCTTTGGCAACGTGGACAGCAGCGGAATCAAGCACAACATTTTCAATCCCCCTATCATTGCTAGATACATCCGGCTGCACCCAACCCATTATTCTATTCGAAGTACACTGAGGATGGAACTGATGGGATGCGATCTGAACAGTTGTTCAATGCCCCTGGGGATGGAGTCCAAGGCAATCTCTGACGCCCAGATTACCGCTAGCTCCTACTTCACTAATATGTTTGCTACCTGGAGCCCTTCCAAAGCAAGACTGCACCTGCAAGGCCGCAGCAACGCATGGCGACCACAGGTGAACAATCCCAAGGAGTGGTTGCAGGTCGATTTTCAGAAAACTATGAAGGTGACCGGGGTCACAACTCAGGGCGTGAAAAGTCTGCTGACCTCAATGTACGTCAAGGAGTTCCTGATCTCTAGTTCACAGGACGGACATCAGTGGACACTGTTCTTTCAGAACGGGAAGGTGAAAGTCTTCCAGGGCAATCAGGATTCCTTTACACCTGTGGTCAACAGTCTAGACCCTCCACTGCTGACCAGATACCTGAGAATCCACCCTCAGTCCTGGGTGCACCAGATTGCCCTGAGAATGGAAGTGCTGGGATGCGAGGCCCAGGATCTGTACTGATAACTCGAGTCGACC optimized BDD FVIII SEQ ID NO: 2GGCGCGCCCGTACGGCCGCCACCATGCAGATCGAGCTGTCTACCTGCTTCTTCCTGTGCCTGCTGCGGTTCTGCTTCAGCGCCACCCGGCGGTACTACCTGGGCGCCGTGGAACTGAGCTGGGACTACATGCAGAGCGACCTGGGGGAGCTGCCCGTGGACGCCAGATTCCCCCCAAGAGTGCCCAAGAGCTTCCCCTTCAACACCTCCGTGGTGTACAAGAAAACCCTGTTCGTCGAGTTCACCGACCACCTGTTCAATATCGCCAAGCCCAGACCCCCCTGGATGGGCCTGCTGGGCCCTACAATCCAGGCCGAGGTGTACGACACCGTGGTCATCACCCTTAAGAACATGGCCAGCCACCCCGTGTCCCTGCACGCCGTGGGCGTGTCCTACTGGAAGGCCTCTGAGGGCGCTGAGTACGACGACCAGACCAGCCAGCGCGAGAAAGAGGACGACAAAGTCTTTCCTGGCGGCAGCCATACCTACGTGTGGCAGGTCCTGAAAGAAAACGGCCCTATGGCCTCCGACCCCCTGTGCCTGACCTACAGCTACCTGAGCCACGTGGACCTGGTCAAGGACCTGAACAGCGGCCTGATTGGCGCCCTGCTCGTGTGTAGAGAGGGCAGCCTCGCCAAAGAGAAAACCCAGACCCTGCACAAGTTCATCCTGCTGTTCGCCGTGTTCGACGAGGGCAAGAGCTGGCACAGCGAGACAAAGAACAGCCTGATGCAGGACCGGGACGCCGCCTCTGCCAGAGCCTGGCCTAAGATGCACACCGTGAACGGCTACGTGAACAGAAGCCTGCCCGGACTGATCGGCTGCCACCGGAAGTCCGTGTACTGGCACGTGATCGGCATGGGCACCACCCCCGAGGTGCACAGCATCTTTCTGGAAGGCCACACCTTCCTCGTGCGGAACCACAGACAGGCCAGCCTGGAAATCAGCCCTATCACCTTCCTGACCGCCCAGACACTGCTGATGGACCTGGGCCAGTTCCTGCTGTTTTGCCACATCAGCAGCCACCAGCACGACGGCATGGAAGCCTACGTGAAGGTGGACAGCTGCCCCGAGGAACCCCAGCTGCGGATGAAGAACAACGAGGAAGCCGAGGACTACGACGACGACCTGACCGACAGCGAGATGGACGTCGTGCGCTTCGACGACGACAACAGCCCCAGCTTCATCCAGATCAGAAGCGTGGCCAAGAAGCACCCCAAGACCTGGGTGCACTATATCGCCGCCGAGGAAGAGGACTGGGACTACGCCCCTCTGGTGCTGGCCCCCGACGACAGAAGCTACAAGAGCCAGTACCTGAACAATGGCCCCCAGCGGATCGGCCGGAAGTACAAGAAAGTGCGGTTCATGGCCTACACCGACGAGACATTCAAGACCAGAGAGGCCATCCAGCACGAGAGCGGCATCCTGGGCCCCCTGCTGTATGGCGAAGTGGGCGACACCCTGCTGATCATCTTCAAGAACCAGGCCAGCCGGCCCTACAACATCTACCCCCACGGCATCACCGACGTGCGGCCCCTGTACAGCAGACGGCTGCCCAAGGGCGTGAAGCACCTGAAGGACTTCCCCATCCTGCCCGGCGAGATCTTCAAGTACAAGTGGACCGTGACCGTGGAAGATGGCCCCACCAAGAGCGACCCCAGATGCCTGACCCGGTACTACAGCAGCTTCGTGAACATGGAACGGGACCTGGCCTCCGGGCTGATCGGCCCTCTGCTGATCTGCTACAAAGAAAGCGTGGACCAGCGGGGCAACCAGATCATGAGCGACAAGCGGAACGTGATCCTGTTCAGCGTGTTCGATGAGAATCGGTCCTGGTACCTGACCGAGAATATCCAGCGGTTCCTGCCCAACCCTGCCGGCGTGCAGCTGGAAGATCCCGAGTTCCAGGCCAGCAACATCATGCACTCCATCAATGGCTACGTGTTCGACAGCCTCCAGCTGAGCGTGTGCCTGCACGAGGTGGCCTACTGGTACATCCTGAGCATCGGCGCCCAGACCGACTTCCTGAGCGTGTTCTTCAGCGGCTACACCTTCAAGCACAAGATGGTGTACGAGGATACCCTGACCCTGTTCCCCTTCTCCGGCGAAACCGTGTTCATGAGCATGGAAAACCCCGGCCTGTGGATTCTGGGCTGCCACAACAGCGACTTCAGAAACCGGGGCATGACCGCCCTGCTGAAGGTGTCCAGCTGCGACAAGAACACCGGCGACTACTACGAGGACAGCTATGAGGACATCAGCGCCTACCTGCTGAGCAAGAACAACGCCATCGAGCCCAGATCCTTCAGCCAGAACCCCCCCGTGCTGAAGCGGCACCAGAGAGAGATCACCCGGACCACCCTGCAGTCCGACCAGGAAGAGATTGATTACGACGACACCATCAGCGTCGAGATGAAGAAAGAGGATTTCGACATCTACGACGAGGACGAGAACCAGAGCCCCCGGTCCTTCCAGAAGAAAACCCGGCACTACTTCATTGCCGCCGTGGAAAGACTGTGGGACTACGGCATGAGCAGCAGCCCCCACGTGCTGCGGAACAGAGCCCAGAGCGGCAGCGTGCCCCAGTTCAAGAAAGTGGTGTTCCAGGAGTTCACCGACGGCAGCTTCACCCAGCCCCTGTATCGGGGCGAGCTGAACGAGCACCTGGGACTGCTGGGACCTTACATTAGAGCCGAGGTGGAAGATAACATCATGGTCACCTTCAGAAACCAGGCCTCCAGACCCTACAGCTTCTACAGCAGCCTGATCAGCTACGAAGAGGACCAGCGGCAGGGCGCCGAACCCCGGAAGAACTTCGTGAAGCCCAACGAGACTAAGACCTACTTCTGGAAGGTGCAGCACCACATGGCCCCCACAAAGGACGAGTTCGACTGCAAGGCCTGGGCCTACTTCTCCGATGTGGACCTGGAAAAGGACGTGCACTCTGGCCTGATTGGACCTCTGCTCGTCTGCCACACCAACACCCTGAACCCCGCCCACGGCCGGCAGGTCACAGTGCAGGAATTTGCCCTGTTCTTCACCATCTTCGATGAGACAAAGAGCTGGTACTTCACCGAGAACATGGAAAGAAACTGTAGAGCCCCCTGCAACATCCAGATGGAAGATCCTACCTTCAAAGAGAACTATCGGTTCCACGCCATCAACGGCTACATCATGGACACCCTGCCCGGCCTGGTCATGGCCCAGGATCAGAGAATCCGGTGGTATCTGCTGAGCATGGGCAGCAACGAGAACATCCACAGCATCCACTTCAGCGGCCACGTGTTCACAGTGCGGAAGAAAGAAGAGTACAAGATGGCCCTGTACAACCTGTACCCCGGCGTGTTCGAGACAGTGGAAATGCTGCCCAGCAAGGCCGGCATCTGGCGGGTGGAATGTCTGATCGGCGAGCATCTGCACGCCGGAATGAGCACCCTGTTTCTGGTGTACAGCAACAAGTGCCAGACCCCTCTGGGCATGGCCAGCGGCCACATCCGGGACTTCCAGATCACCGCCTCCGGCCAGTACGGCCAGTGGGCCCCTAAGCTGGCCCGGCTCCACTACTCCGGATCTATCAACGCCTGGTCCACCAAAGAGCCCTTCAGCTGGATCAAGGTGGACCTGCTGGCCCCTATGATCATCCACGGAATCAAGACCCAGGGCGCCAGACAGAAGTTCAGCAGCCTGTACATCAGCCAGTTCATCATCATGTACAGCCTGGACGGCAAGAAGTGGCAGACCTACCGGGGCAACAGCACCGGCACCCTGATGGTGTTCTTCGGCAACGTGGACAGCAGCGGCATCAAGCACAACATCTTCAACCCCCCCATCATTGCCCGGTACATCCGGCTGCACCCCACCCACTACAGCATCCGGTCCACCCTGCGGATGGAACTGATGGGCTGCGACCTGAACTCTTGCAGCATGCCCCTGGGGATGGAAAGCAAGGCCATCAGCGACGCCCAGATCACAGCGAGCAGCTACTTCACCAACATGTTCGCCACCTGGTCCCCAAGCAAAGCCCGCCTGCATCTCCAAGGCAGAAGCAATGCCTGGCGGCCTCAGGTCAACAACCCCAAAGAATGGCTCCAGGTGGACTTTCAGAAAACCATGAAGGTCACAGGCGTGACCACCCAGGGCGTGAAAAGCCTGCTGACCTCTATGTACGTGAAAGAGTTCCTGATCAGCAGCAGCCAGGACGGGCACCAGTGGACCCTGTTCTTTCAGAACGGCAAAGTGAAAGTGTTCCAGGGCAACCAGGACTCCTTTACCCCCGTGGTCAACTCTCTAGACCCTCCACTGCTGACCAGATACCTGAGAATCCACCCTCAGTCCTGGGTGCACCAGATTGCCCTGAGAATGGAAGTGCTGGGATGCGAGGCCCAGGATCTGTACTGATAACTCGAGTCGACTTAATTAA Nucleotide Sequence Encoding “Parental” BDD FVIIISEQ ID NO: 3ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTGCTTTAGTGCCACCAGAAGATACTACCTGGGTGCAGTGGAACTGTCATGGGACTATATGCAAAGTGATCTCGGTGAGCTGCCTGTGGACGCAAGATTTCCTCCTAGAGTGCCAAAATCTTTTCCATTCAACACCTCAGTCGTGTACAAAAAGACTCTGTTTGTAGAATTCACGGATCACCTTTTCAACATCGCTAAGCCAAGGCCACCCTGGATGGGTCTGCTAGGTCCTACCATCCAGGCTGAGGTTTATGATACAGTGGTCATTACACTTAAGAACATGGCTTCCCATCCTGTCAGTCTTCATGCTGTTGGTGTATCCTACTGGAAAGCTTCTGAGGGAGCTGAATATGATGATCAGACCAGTCAAAGGGAGAAAGAAGATGATAAAGTCTTCCCTGGTGGAAGCCATACATATGTCTGGCAGGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCACTGTGCCTTACCTACTCATATCTTTCTCATGTGGACCTGGTAAAAGACTTGAATTCAGGCCTCATTGGAGCCCTACTAGTATGTAGAGAAGGGAGTCTGGCCAAGGAAAAGACACAGACCTTGCACAAATTTATACTACTTTTTGCTGTATTTGATGAAGGGAAAAGTTGGGACTCAGAAACAAAGAACTCCTTGATGCAGGATAGGGATGCTGCATCTGCTCGGGCCTGGCCTAAAATGCACACAGTCAATGGTTATGTAAACAGGTCTCTGCCAGGTCTGATTGGATGCCACAGGAAATCAGTCTATTGGCATGTGATTGGAATGGGCACCACTCCTGAAGTGCACTCAATATTCCTCGAAGGTCACACATTTCTTGTGAGGAACCATCGCCAGGCGTCCTTGGAAATCTCGCCAATAACTTTCCTTACTGCTCAAACACTCTTGATGGACCTTGGACAGTTTCTACTGTTTTGTCATATCTCTTCCCACCAACATGATGGCATGGAAGCTTATGTCAAAGTAGACAGCTGTCCAGAGGAACCCCAACTACGAATGAAAAATAATGAAGAAGCGGAAGACTATGATGATGATCTTACTGATTCTGAAATGGATGTGGTCAGGTTTGATGATGACAACTCTCCTTCCTTTATCCAAATTCGCTCAGTTGCCAAGAAGCATCCTAAAACTTGGGTACATTACATTGCTGCTGAAGAGGAGGACTGGGACTATGCTCCCTTAGTCCTCGCCCCCGATGACAGAAGTTATAAAAGTCAATATTTGAACAATGGCCCTCAGCGGATTGGTAGGAAGTACAAAAAAGTCCGATTTATGGCATACACAGATGAAACCTTTAAGACTCGTGAAGCTATTCAGCATGAATCAGGAATCTTGGGACCTTTACTTTATGGGGAAGTTGGAGACACACTGTTGATTATATTTAAGAATCAAGCAAGCAGACCATATAACATCTACCCTCACGGAATCACTGATGTCCGTCCTTTGTATTCAAGGAGATTACCAAAAGGTGTAAAACATTTGAAGGATTTTCCAATTCTGCCAGGAGAAATATTCAAATATAAATGGACAGTGACTGTAGAAGATGGGCCAACTAAATCAGATCCTCGGTGCCTGACCCGCTATTACTCTAGTTTCGTTAATATGGAGAGAGATCTAGCTTCAGGACTCATTGGCCCTCTCCTCATCTGCTACAAAGAATCTGTAGATCAAAGAGGAAACCAGATAATGTCAGACAAGAGGAATGTCATCCTGTTTTCTGTATTTGATGAGAACCGAAGCTGGTACCTCACAGAGAATATACAACGCTTTCTCCCCAATCCAGCTGGAGTGCAGCTTGAGGATCCAGAGTTCCAAGCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGTTTGGAGTTGTCAGTTTGTTTGCATGAGGTGGCATACTGGTACATTCTAAGCATTGGAGCACAGACTGACTTCCTTTCTGTCTTCTTCTCTGGATATACCTTCAAACACAAAATGGTCTATGAAGACACACTCACCCTATTCCCATTCTCAGGAGAAACTGTCTTCATGTCGATGGAAAACCCAGGTCTATGGATTCTGGGGTGCCACAACTCAGACTTTCGGAACAGAGGCATGACCGCCTTACTGAAGGTTTCTAGTTGTGACAAGAACACTGGTGATTATTACGAGGACAGTTATGAAGATATTTCAGCATACTTGCTGAGTAAAAACAATGCCATTGAACCAAGAAGCTTCTCTCAAAACCCACCAGTCTTGAAACGCCATCAACGGGAAATAACTCGTACTACTCTTCAGTCAGATCAAGAGGAAATTGACTATGATGATACCATATCAGTTGAAATGAAGAAGGAAGATTTTGACATTTATGATGAGGATGAAAATCAGAGCCCCCGCAGCTTTCAAAAGAAAACACGACACTATTTTATTGCTGCAGTGGAGAGGCTCTGGGATTATGGGATGAGTAGGTCCCCACATGTTGTAAGAAACAGGGCTCAGAGTGGGAGTGTGGCTCAGTTCAAGAAAGTTGTTTTCCAGGAATTTACTGATGGCTCCTTTACTCAGCCCTTATACCGTGGAGAACTAAATGAACATTTGGGACTCCTGGGGCCATATATAAGAGCAGAAGTTGAAGATAATATCATGGTAACTTTCAGAAATCAGGCCTCTCGTCCCTATTCCTTCTATTCTAGCCTTATTTCTTATGAGGAAGATCAGAGGCAAGGAGCAGAACCTAGAAAAAACTTTGTCAAGCCTAATGAAACCAAAACTTACTTTTGGAAAGTGCAACATCATATGGCACCCACTAAAGATGAGTTTGACTGCAAAGCCTGGGCTTATTTCTCTGATGTTGACCTGGAAAAAGATGTGCACTCAGGCCTGATTGGACCCCTTCTGGTCTGCCACACTAACACACTGAACCCTGCTCATGGGAGACAAGTGACAGTACAGGAATTTGCTCTGTTTTTCACCATCTTTGATGAGACCAAAAGCTGGTACTTCACTGAAAATATGGAAAGAAACTGCAGGGCTCCCTGCAATATCCAGATGGAAGATCCCACTTTTAAAGAGAATTATCGCTTCCATGCAATCAATGGCTACATAATGGATACACTACCTGGCTTAGTAATGGCTCAGGATCAAAGGATTCGATGGTATCTGCTCAGCATGGGCAGCAATGAAAACATCCATTCTATTCATTTCAGTGGACATGTGTTCACTGTACGAAAAAAAGAGGAGTATAAAATGGCACTGTACAATCTCTATCCAGGTGTTTTTGAGACAGTGGAAATGTTACCATCCAAAGCTGGAATTTGGCGGGTGGAATGCCTTATTGGCGAGCATCTACATGCTGGGATGAGCACACTTTTTCTGGTGTACAGCAATAAGTGTCAGACTCCCCTGGGAATGGCTTCTGGACACATTAGAGATTTTCAGATTACAGCTTCAGGACAATATGGACAGTGGGCCCCAAAGCTGGCCAGACTTCATTATTCCGGATCAATCAATGCCTGGAGCACCAAGGAGCCCTTTTCTTGGATCAAGGTGGATCTGTTGGCACCAATGATTATTCACGGCATCAAGACCCAGGGTGCCCGTCAGAAGTTCTCCAGCCTCTACATCTCTCAGTTTATCATCATGTATAGTCTTGATGGGAAGAAGTGGCAGACTTATCGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAATGTGGATTCATCTGGGATAAAACACAATATTTTTAACCCTCCAATTATTGCTCGATACATCCGTTTGCACCCAACTCATTATAGCATTCGCAGCACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAATAGTTGCAGCATGCCATTGGGAATGGAGAGTAAAGCAATATCAGATGCACAGATTACTGCTTCATCCTACTTTACCAATATGTTTGCCACCTGGTCTCCTTCAAAAGCTCGACTTCACCTCCAAGGGAGGAGTAATGCCTGGAGACCTCAGGTGAATAATCCAAAAGAGTGGCTGCAAGTGGACTTCCAGAAGACAATGAAAGTCACAGGAGTAACTACTCAGGGAGTAAAATCTCTGCTTACCAGCATGTATGTGAAGGAGTTCCTCATCTCCAGCAGTCAAGATGGCCATCAGTGGACTCTCTTTTTTCAGAATGGCAAAGTAAAGGTTTTTCAGGGAAATCAAGACTCCTTCACACCTGTGGTGAACTCTCTAGACCCACCGTTACTGACTCGCTACCTTCGAATTCACCCCCAGAGTTGGGTGCACCAGATTGCCCTGAGGATGGAGGTTCTGGGCTGCGAGGCACAGGACCTCTAC Amino Acid Sequenceof BDD FVIII SEQ ID NO: 4ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEWYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTRSAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYF1AAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQTTASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLTSSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYMAR/ARS nucleotide sequence SEQ ID NO: 5 ATATTT MAR/ARS nucleotidesequence SEQ ID NO: 6 AAATAT Potential Splice Site SEQ ID NO: 7 GGTGATDestabilizing Sequence SEQ ID NO: 8 ATTTA Destabilizing Sequence SEQ IDNO: 9 TAAAT poly-T Sequence SEQ ID NO: 10 TTTTTT poly-A Sequence SEQ IDNO: 11 AAAAAAA Promoter Binding Site SEQ ID NO: 12 TATAA PromoterBinding Site SEQ ID NO: 13 TTATA AU Rich Sequence Elements (ARE) SEQ IDNO: 14 ATTTTATT AU Rich Sequence Elements (ARE) SEQ ID NO: 15 ATTTTTAAKozak Consensus Sequence SEQ ID NO: 16 GCCGCCACCATGC CTP peptide SEQ IDNO: 17 DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL CTP peptide SEQ ID NO: 18SSSSKAPPPSLPSPSRLPGPSDTPILPQ albumin-binding peptides core sequence SEQID NO: 19 DICLPRWGCLW PAS Sequence SEQ ID NO: 20 ASPAAPAPASPAAPAPSAPAPAS Sequence SEQ ID NO: 21 AAPASPAPAAPSAPAPAAPS PAS Sequence SEQ ID NO:22 APSSPSPSAPSSPSPASPSS PAS Sequence SEQ ID NO: 23 APSSPSPSAPSSPSPASPSPAS Sequence SEQ ID NO: 24 SSPSAPSPSSPASPSPSSPA PAS Sequence SEQ ID NO:25 AASPAAPSAPPAAASPAAPSAPPA PAS Sequence SEQ ID NO: 26ASAAAPAAASAAASAPSAAA Full length von Willebrand Factor nucleotidesequence SEQ ID NO: 27ATGATTCCTGCCAGATTTGCCGGGGTGCTGCTTGCTCTGGCCCTCATTTTGCCAGGGACCCTTTGTGCAGAAGGAACTCGCGGCAGGTCATCCACGGCCCTACTAAGGACGGTCTAAACGGCCCCACGACGAACGAGACCGGGAGTAAAACGGTCCCTGGGAAACACGTCTTCCTTGAGCGCCGTCCAGTAGGTGCCGGGGATGCAGCCTTTTCGGAAGTGACTTCGTCAACACCTTTGATGGGAGCATGTACAGCTTTGCGGGATACTGCAGTTACCTCCTGGCAGGGGGCTGCCAGAACTACGTCGGAAAAGCCTTCACTGAAGCAGTTGTGGAAACTACCCTCGTACATGTCGAAACGCCCTATGACGTCAATGGAGGACCGTCCCCCGACGGTCTTACGCTCCTTCTCGATTATTGGGGACTTCCAGAATGGCAAGAGAGTGAGCCTCTCCGTGTATCTTGGGGAATTTTTTGACATCCATTTGTTTGTCAATGGTTGCGAGGAAGAGCTAATAACCCCTGAAGGTCTTACCGTTCTCTCACTCGGAGAGGCACATAGAACCCCTTAAAAAACTGTAGGTAAACAAACAGTTACCAACCGTGACACAGGGGGACCAAAGAGTCTCCATGCCCTATGCCTCCAAAGGGCTGTATCTAGAAACTGAGGCTGGGTACTACAAGCTGTCCGGTGAGGCCTTGGCACTGTGTCCCCCTGGTTTCTCAGAGGTACGGGATACGGAGGTTTCCCGACATAGATCTTTGACTCCGACCCATGATGTTCGACAGGCCACTCCGGAATGGCTTTGTGGCCAGGATCGATGGCAGCGGCAACTTTCAAGTCCTGCTGTCAGACAGATAGTTCAACAAGACCTGCGGGCTGTGTGGCAACTTTAACATTACCGAAACACCGGTCCTAGCTACCGTCGCCGTTGAAAGTTCAGGACGACAGTCTGTCTATGAAGTTGTTCTGGACGCCCGACACACCGTTGAAATTGTACTTTGCTGAAGATGACTTTATGACCCAAGAAGGGACCTTGACCTCGGACCCTTATGACTTTGCCAACTCATGGGCTCTGAGCAGTGGAGAACAGTGGTGTGAAACGACTTCTACTGAAATACTGGGTTCTTCCCTGGAACTGGAGCCTGGGAATACTGAAACGGTTGAGTACCCGAGACTCGTCACCTCTTGTCACCACAGAACGGGCATCTCCTCCCAGCAGCTCATGCAACATCTCCTCTGGGGAAATGCAGAAGGGCCTGTGGGAGCAGTGCCAGCTTCTGAAGAGCACCTCGGTGTCTTGCCCGTAGAGGAGGGTCGTCGAGTACGTTGTAGAGGAGACCCCTTTACGTCTTCCCGGACACCCTCGTCACGGTCGAAGACTTCTCGTGGAGCCACATTGCCCGCTGCCACCCTCTGGTGGACCCCGAGCCTTTTGTGGCCCTGTGTGAGAAGACTTTGTGTGAGTGTGCTGGGGGGCTGGAGTGCGCCTGCCCTGCAACGGGCGACGGTGGGAGACCACCTGGGGCTCGGAAAACACCGGGACACACTCTTCTGAAACACACTCACACGACCCCCCGACCTCACGCGGACGGGACGCCTCCTGGAGTACGCCCGGACCTGTGCCCAGGAGGGAATGGTGCTGTACGGCTGGACCGACCACAGCGCGTGCAGCCCAGTGTGCCCTGCTGGTATGGAGGGAGGACCTCATGCGGGCCTGGACACGGGTCCTCCCTTACCACGACATGCCGACCTGGCTGGTGTCGCGCACGTCGGGTCACACGGGACGACCATACCTCTATAGGCAGTGTGTGTCCCCTTGCGCCAGGACCTGCCAGAGCCTGCACATCAATGAAATGTGTCAGGAGCGATGCGTGGATGGCTGCAGCTGCCCTGAGGATATCCGTCACACACAGGGGAACGCGGTCCTGGACGGTCTCGGACGTGTAGTTACTTTACACAGTCCTCGCTACGCACCTACCGACGTCGACGGGACTCCGACAGCTCCTGGATGAAGGCCTCTGCGTGGAGAGCACCGAGTGTCCCTGCGTGCATTCCGGAAAGCGCTACCCTCCCGGCACCTCCCTCTCTCGAGACTGCTGTCGAGGACCTACTTCCGGAGACGCACCTCTCGTGGCTCACAGGGACGCACGTAAGGCCTTTCGCGATGGGAGGGCCGTGGAGGGAGAGAGCTCTGACCAACACCTGCATTTGCCGAAACAGCCAGTGGATCTGCAGCAATGAAGAATGTCCAGGGGAGTGCCTTGTCACTGGTCAATCCCACTTCAAGAGCTTTGACGTTGTGGACGTAAACGGCTTTGTCGGTCACCTAGACGTCGTTACTTCTTACAGGTCCCCTCACGGAACAGTGACCAGTTAGGGTGAAGTTCTCGAAACTGAACAGATACTTCACCTTCAGTGGGATCTGCCAGTACCTGCTGGCCCGGGATTGCCAGGACCACTCCTTCTCCATTGTCATTGAGACTGTCCAGTGTGCTGTTGTCTATGAAGTGGAAGTCACCCTAGACGGTCATGGACGACCGGGCCCTAACGGTCCTGGTGAGGAAGAGGTAACAGTAACTCTGACAGGTCACACGACATGACCGCGACGCTGTGTGCACCCGCTCCGTCACCGTCCGGCTGCCTGGCCTGCACAACAGCCTTGTGAAACTGAAGCATGGGGCAGGAGTTGCCATGGATACTGGCGCTGCGACACACGTGGGCGAGGCAGTGGCAGGCCGACGGACCGGACGTGTTGTCGGAACACTTTGACTTCGTACCCCGTCCTCAACGGTACCTTGGCCAGGACATCCAGCTCCCCCTCCTGAAAGGTGACCTCCGCATCCAGCATACAGTGACGGCCTCCGTGCGCCTCAGCTACGGGGAGGACCTGCAGATGACCGGTCCTGTAGGTCGAGGGGGAGGACTTTCCACTGGAGGCGTAGGTCGTATGTCACTGCCGGAGGCACGCGGAGTCGATGCCCCTCCTGGACGTCTACGACTGGGATGGCCGCGGGAGGCTGCTGGTGAAGCTGTCCCCCGTCTATGCCGGGAAGACCTGCGGCCTGTGTGGGAATTACAATGGCAACCAGGGCGACGCTGACCCTACCGGCGCCCTCCGACGACCACTTCGACAGGGGGCAGATACGGCCCTTCTGGACGCCGGACACACCCTTAATGTTACCGTTGGTCCCGCTGCACTTCCTTACCCCCTCTGGGCTGGCRGAGCCCCGGGTGGAGGACTTCGGGAACGCCTGGAAGCTGCACGGGGACTGCCAGGACCTGCAGAAGCAGCACAGTGAAGGAATGGGGGAGACCCGACCGYCTCGGGGCCCACCTCCTGAAGCCCTTGCGGACCTTCGACGTGCCCCTGACGGTCCTGGACGTCTTCGTCGTGTCCGATCCCTGCGCCCTCAACCCGCGCATGACCAGGTTCTCCGAGGAGGCGTGCGCGGTCCTGACGTCCCCCACATTCGAGGCCTGCCATCGTGCCGTCAGCGCTAGGGACGCGGGAGTTGGGCGCGTACTGGTCCAAGAGGCTCCTCCGCACGCGCCAGGACTGCAGGGGGTGTAAGCTCCGGACGGTAGCACGGCAGTCGCCGCTGCCCTACCTGCGGAACTGCCGCTACGACGTGTGCTCCTGCTCGGACGGCCGCGAGTGCCTGTGCGGCGCCCTGGCCAGCTATGCCGCGGCCTGCGGGCGACGGGATGGACGCCTTGACGGCGATGCTGCACACGAGGACGAGCCTGCCGGCGCTCACGGACACGCCGCGGGACCGGTCGATACGGCGCCGGACGCCGGGGAGAGGCGTGCGCGTCGCGTGGCGCGAGCCAGGCCGCTGTGAGCTGAACTGCCCGAAAGGCCAGGTGTACCTGCAGTGCGGGACCCCCTGCAACCTGCCCCTCTCCGCACGCGCAGCGCACCGCGCTCGGTCCGGCGACACTCGACTTGACGGGCTTTCCGGTCCACATGGACGTCACGCCCTGGGGGACGTTGGAGACCTGCCGCTCTCTCTCTTACCCGGATGAGGAATGCAATGAGGCCTGCCTGGAGGGCTGCTTCTGCCCCCCAGGGCTCTACATGGATGAGAGGGGGGACCTGGACGGCGAGAGAGAGAATGGGCCTACTCCTTACGTTACTCCGGACGGACCTCCCGACGAAGACGGGGGGTCCCGAGATGTACCTACTCTCCCCCCTGTGCGTGCCCAAGGCCCAGTGCCCCTGTTACTATGACGGTGAGATCTTCCAGCCAGAAGACATCTTCTCAGACCATCACACCATGTGCTACTGTGAGGATGACGCACGGGTTCCGGGTCACGGGGACAATGATACTGCCACTCTAGAAGGTCGGTCTTCTGTAGAAGAGTCTGGTAGTGTGGTACACGATGACACTCCTACGCTTCATGCACTGTACCATGAGTGGAGTCCCCGGAAGCTTGCTGCCTGACGCTGTCCTCAGCAGTCCCCTGTCTCATCGCAGCAAAAGGAGCCTATCCTGCGAAGTACGTGACATGGTACTCACCTCAGGGGCCTTCGAACGACGGACTGCGACAGGAGTCGTCAGGGGACAGAGTAGCGTCGTTTTCCTCGGATAGGACTCGGCCCCCCATGGTCAAGCTGGTGTGTCCCGCTGACAACCTGCGGGCTGAAGGGCTCGAGTGTACCAAAACGTGCCAGAACTATGACCTGGAGTGCATGAGCCGGGGGGTACCAGTTCGACCACACAGGGCGACTGTTGGACGCCCGACTTCCCGAGCTCACATGGTTTTGCACGGTCTTGATACTGGACCTCACGTACAGCATGGGCTGTGTCTCTGGCTGCCTCTGCCCCCCGGGCATGGTCCGGCATGAGAACAGATGTGTGGCCCTGGAAAGGTGTCCCTGCTTCCATCAGGGCATCGTACCCGACACAGAGACCGACGGAGACGGGGGGCCCGTACCAGGCCGTACTCTTGTCTACACACCGGGACCTTTCCACAGGGACGAAGGTAGTCCCGTAGGAGTATGCCCCTGGAGAAACAGTGAAGATTGGCTGCAACACTTGTGTCTGTCGGGACCGGAAGTGGAACTGCACAGACCATGTGTGTGATGCCACGTGTCCTCATACGGGGACCTCTTTGTCACTTCTAACCGACGTTGTGAACACAGACAGCCCTGGCCTTCACCTTGAGGTGTCTGGTACACACACTACGGTGCACCTCCACGATCGGCATGGCCCACTACCTCACCTTCGACGGGCTCAAATACCTGTTCCCCGGGGAGTGCCAGTACGTTCTGGTGCAGGATTACTGCGGCAGTGAGGTGCTAGCCGTACCGGGTGATGGAGTGGAAGCTGCCCGAGTTTATGGACAAGGGGCCCCTCACGGTCATGCAAGACCACGTCCTAATGACGCCGTCAAACCCTGGGACCTTTCGGATCCTAGTGGGGAATAAGGGATGCAGCCACCCCTCAGTGAAATGCAAGAAACGGGTCACCATCCTGGTGGAGGGAGGAGAGATTGGGACCCTGGAAAGCCTAGGATCACCCCTTATTCCCTACGTCGGTGGGGAGTCACTTTACGTTCTTTGCCCAGTGGTAGGACCACCTCCCTCCTCTCTTTGAGCTGTTTGACGGGGAGGTGAATGTGAAGAGGCCCATGAAGGATGAGACTCACTTTGAGGTGGTGGAGTCTGGCCGGTACATCATTCTGCTGCTGGGAACTCGACAAACTGCCCCTCCACTTACACTTCTCCGGGTACTTCCTACTCTGAGTGAAACTCCACCACCTCAGACCGGCCATGTAGTAAGACGACGACCCCAAAGCCCTCTCCGTGGTCTGGGACCGCCACCTGAGCATCTCCGTGGTCCTGAAGCAGACATACCAGGAGAAAGTGTGTGGCCTGTGTGGGAATTTTGATGTTTCGGGAGAGGCACCAGACCCTGGCGGTGGACTCGTAGAGGCACCAGGACTTCGTCTGTATGGTCCTCTTTCACACACCGGACACACCCTTAAAACTAGGCATCCAGAACAATGACCTCACCAGCAGCAACCTCCAAGTGGAGGAAGACCCTGTGGACTTTGGGAACTCCTGGAAAGTGAGCTCGCAGTGTGCTGACACCGTAGGTCTTGTTACTGGAGTGGTCGTCGTTGGAGGTTCACCTCCTTCTGGGACACCTGAAACCCTTGAGGACCTTTCACTCGAGCGTCACACGACTGTCCAGAAAAGTGCCTCTGGACTCATCCCCTGCCACCTGCCATAACAACATCATGAAGCAGACGATGGTGGATTCCTCCTGTAGAATCCTTACCAGTGACGTGGTCTTTTCACGGAGACCTGAGTAGGGGACGGTGGACGGTATTGTTGTAGTACTTCGTCTGCTACCACCTAAGGAGGACATCTTAGGAATGGTCACTGCACTTCCAGGACTGCAACAAGCTGGTGGACCCCGAGCCATATCTGGATGTCTGCATTTACGACACCTGCTCCTGTGAGTCCATTGGGGACTGCGCCTGCTTCGAAGGTCCTGACGTTGTTCGAGCACCTGGGGCTCGGTATAGACCTACAGACGTAAATGCTGTGGACGAGGACACTCAGGTAACCCCTGACGCGGACGAAGTGCGACACCATTGCTGCCTATGCCCACGTGTGTGCCCAGCATGGCAAGGTGGTGACCTGGAGGACGGCCACATTGTGCCCCCAGAGCTGCGAGGAGAGGAACGCTGTGGTAACGACGGATACGGGTGCACACACGGGTCGTACCGTTCCACCACTGGACCTCCTGCCGGTGTAACACGGGGGTCTCGACGCTCCTCTCCTATCTCCGGGAGAACGGGTATGAGTGTGAGTGGCGCTATAACAGCTGTGCACCTGCCTGTCAAGTCACGTGTCAGCACCCTGAGCCACTGGCCTGCCCTGTTAGAGGCCCTCTTGCCCATACTCACACTCACCGCGATATTGTCGACACGTGGACGGACAGTTCAGTGCACAGTCGTGGGACTCGGTGACCGGACGGGACAGCAGTGTGTGGAGGGCTGCCATGCCCACTGCCCTCCAGGGAAAATCCTGGATGAGCTTTTGCAGACCTGCGTTGACCCTGAAGACTGTCCAGTGTGTGAGCGTCACACACCTCCCGACGGTACGGGTGACGGGAGGTCCCTTTTAGGACCTACTCGAAAACGTCTGGACGCAACTGGGACTTCTGACAGGTCACACACTCGTGGCTGGCCGGCGTTTTGCCTCAGGAAAGAAAGTCACCTTGAATCCCAGTGACCCTGAGCACTGCCAGATTTGCCACTGTGATGTTGTCAACCTCACCTCACCGACCGGCCGCAAAACGGAGTCCTTTCTTTCAGTGGAACTTAGGGTCACTGGGACTCGTGACGGTCTAAACGGTGACACTACAACAGTTGGAGTGGAGTGAAGCCTGCCAGGAGCCGGGAGGCCTGGTGGTGCCTCCCACAGATGCCCCGGTGAGCCCCACCACTCTGTATGTGGAGGACATCTCGGAACCGCCGTTCACTTCGGACGGTCCTCGGCCCTCCGGACCACCACGGAGGGTGTCTACGGGGCCACTCGGGGTGGTGAGACATACACCTCCTGTAGAGCCTTGGCGGCAAGCACGATTTCTACTGCAGCAGGCTACTGGACCTGGTCTTCCTGCTGGATGGCTCCTCCAGGCTGTCCGAGGCTGAGTTTGAAGTGCTGAAGGCCTTTGTGCGTGCTAAAGATGACGTCGTCCGATGACCTGGACCAGAAGGACGACCTACCGAGGAGGTCCGACAGGCTCCGACTCAAACTTCACGACTTCCGGAAACACGTGGACATGATGGAGCGGCTGCGCATCTCCCAGAAGTGGGTCCGCGTGGCCGTGGTGGAGTACCACGACGGCTCCCACGCCTACATCGGGCTCAAGGACCCACCTGTACTACCTCGCCGACGCGTAGAGGGTCTTCACCCAGGCGCACCGGCACCACCTCATGGTGCTGCCGAGGGTGCGGATGTAGCCCGAGTTCCTGGGGAAGCGACCGTCAGAGCTGCGGCGCATTGCCAGCCAGGTGAAGTATGCGGGCAGCCAGGTGGCCTCCACCAGCGAGGTCTTGAAATACACACTGTTCCACCTTCGCTGGCAGTCTCGACGCCGCGTAACGGTCGGTCCACTTCATACGCCCGTCGGTCCACCGGAGGTGGTCGCTCCAGAACTTTATGTGTGACAAGGTAATCTTCAGCAAGATCGACCGCCCTGAAGCCTCCCGCATCGCCCTGCTCCTGATGGCCAGCCAGGAGCCCCAACGGATGTCCCGGAACTTTGTCCGCTACTTAGAAGTCGTTCTAGCTGGCGGGACTTCGGAGGGCGTAGCGGGACGAGGACTACCGGTCGGTCCTCGGGGTTGCCTACAGGGCCTTGAAACAGGCGATGGTCCAGGGCCTGAAGAAGAAGAAGGTCATTGTGATCCCGGTGGGCATTGGGCCCCATGCCAACCTCAAGCAGATCCGCCTCATCGAGAAGCAGGCCCCTGCAGGTCCCGGACTTCTTCTTCTTCCAGTAACACTAGGGCCACCCGTAACCCGGGGTACGGTTGGAGTTCGTCTAGGCGGAGTAGCTCTTCGTCCGGGGACAGAACAAGGCCTTCGTGCTGAGCAGTGTGGATGAGCTGGAGCAGCAAAGGGACGAGATCGTTAGCTACCTCTGTGACCTTGCCCCTGAAGCCCCTCCTCCTCTTGTTCCGGAAGCACGACTCGTCACACCTACTCGACCTCGTCGTTTCCCTGCTCTAGCAATCGATGGAGACACTGGAACGGGGACTTCGGGGAGGAGGTACTCTGCCCCCCGACATGGCACAAGTCACTGTGGGCCCGGGGCTCTTGGGGGTTTCGACCCTGGGGCCCAAGAGGAACTCCATGGTTCTGGATGTGGCGATGAGACGGGGGGCTGTACCGTGTTCAGTGACACCCGGGCCCCGAGAACCCCCAAAGCTGGGACCCCGGGTTCTCCTTGAGGTACCAAGACCTACACCGCTTCGTCCTGGAAGGATCGGACAAAATTGGTGAAGCCGACTTCAACAGGAGCAAGGAGTTCATGGAGGAGGTGATTCAGCGGATGGATGTGGGCCAGGACAAAGCAGGACCTTCCTAGCCTGTTTTAACCACTTCGGCTGAAGTTGTCCTCGTTCCTCAAGTACCTCCTCCACTAAGTCGCCTACCTACACCCGGTCCTGTGCATCCACGTCACGGTGCTGCAGTACTCCTACATGGTGACCGTGGAGTACCCCTTCAGCGAGGCACAGTCCAAAGGGGACATCCTGCAGCGGGTGCGAGACGTAGGTGCAGTGCCACGACGTCATGAGGATGTACCACTGGCACCTCATGGGGAAGTCGCTCCGTGTCAGGTTTCCCCTGTAGGACGTCGCCCACGCTCTGATCCGCTACCAGGGCGGCAACAGGACCAACACTGGGCTGGCCCTGCGGTACCTCTCTGACCACAGCTTCTTGGTCAGCCAGGGTGACCGGGAGCAGGCGCTAGGCGATGGTCCCGCCGTTGTCCTGGTTGTGACCCGACCGGGACGCCATGGAGAGACTGGTGTCGAAGAACCAGTCGGTCCCACTGGCCCTCGTCCGCCCCAACCTGGTCTACATGGTCACCGGAAATCCTGCCTCTGATGAGATCAAGAGGCTGCCTGGAGACATCCAGGTGGTGCCCATTGGAGTGGGCCCTAATGGGGTTGGACCAGATGTACCAGTGGCCTTTAGGACGGAGACTACTCTAGTTCTCCGACGGACCTCTGTAGGTCCACCACGGGTAACCTCACCCGGGATTACCCAACGTGCAGGAGCTGGAGAGGATTGGCTGGCCCAATGCCCCTATCCTCATCCAGGACTTTGAGACGCTCCCCCGAGAGGCTCCTGACCTGGTGCTGCAGGTTGCACGTCCTCGACCTCTCCTAACCGACCGGGTTACGGGGATAGGAGTAGGTCCTGAAACTCTGCGAGGGGGCTCTCCGAGGACTGGACCACGACGTGAGGTGCTGCTCCGGAGAGGGGCTGCAGATCCCCACCCTCTCCCCTGCACCTGACTGCAGCCAGCCCCTGGACGTGATCCTTCTCCTGGATGGCTCCTCCCTCCACGACGAGGCCTCTCCCCGACGTCTAGGGGTGGGAGAGGGGACGTGGACTGACGTCGGTCGGGGACCTGCACTAGGAAGAGGACCTACCGAGGAGGAGTTTCCCAGCTTCTTATTTTGATGAAATGAAGAGTTTCGCCAAGGCTTTCATTTCAAAAGCCAATATAGGGCCTCGTCTCACTCAGGTGTCAGTGCTGCTCAAAGGGTCGAAGAATAAAACTACTTTACTTCTCAAAGCGGTTCCGAAAGTAAAGTTTTCGGTTATATCCCGGAGCAGAGTGAGTCCACAGTCACGACGAGTATGGAAGCATCACCACCATTGACGTGCCATGGAACGTGGTCCCGGAGAAAGCCCATTTGCTGAGCCTTGTGGACGTCATGCAGCGGGAGGGAGGCCCTCATACCTTCGTAGTGGTGGTAACTGCACGGTACCTTGCACCAGGGCCTCTTTCGGGTAAACGACTCGGAACACCTGCAGTACGTCGCCCTCCCTCCGGGCAGCCAAATCGGGGATGCCTTGGGCTTTGCTGTGCGATACTTGACTTCAGAAATGCATGGTGCCAGGCCGGGAGCCTCAAAGGCGGTGGTCATCCTGGTCGTCGGTTTAGCCCCTACGGAACCCGAAACGACACGCTATGAACTGAAGTCTTTACGTACCACGGTCCGGCCCTCGGAGTTTCCGCCACCAGTAGGACCAGACGGACGTCTCTGTGGATTCAGTGGATGCAGCAGCTGATGCCGCCAGGTCCAACAGAGTGACAGTGTTCCCTATTGGAATTGGAGATCGCTACGATGCAGTGCCTGCAGAGACACCTAAGTCACCTACGTCGTCGACTACGGCGGTCCAGGTTGTCTCACTGTCACAAGGGATAACCTTAACCTCTAGCGATGCTACGTCCCCAGCTACGGATCTTGGCAGGCCCAGCAGGCGACTCCAACGTGGTGAAGCTCCAGCGAATCGAAGACCTCCGTACCATGGTCACCTTGGGCAATTCCTTGGGTCGATGCCTAGAACCGTCCGGGTCGTCCGCTGAGGTTGCACCACTTCGAGGTCGCTTAGCTTCTGGAGGGATGGTACCAGTGGAACCCGTTAAGGAACCTCCACAAACTGTGCTCTGGATTTGTTAGGATTTGCATGGATGAGGATGGGAATGAGAAGAGGCCCGGGGACGTCTGGACCTTGCCAGACCAGTGCCACGGAGGTGTTTGACACGAGACCTAAACAATCCTAAACGTACCTACTCCTACCCTTACTCTTCTCCGGGCCCCTGCAGACCTGGAACGGTCTGGTCACGGTGACCGTGACTTGCCAGCCAGATGGCCAGACCTTGCTGAAGAGTCATCGGGTCAACTGTGACCGGGGGCTGAGGCCTTCGTGCCCTAACAGCCAGTCCCCTGTGGCACTGAACGGTCGGTCTACCGGTCTGGAACGACTTCTCAGTAGCCCAGTTGACACTGGCCCCCGACTCCGGAAGCACGGGATTGTCGGTCAGGGGACTTAAAGTGGAAGAGACCTGTGGCTGCCGCTGGACCTGCCCCTGYGTGTGCACAGGCAGCTCCACTCGGCACATCGTGACCTTTGATGGGCAGAATTTCAAAATTTCACCTTCTCTGGACACCGACGGCGACCTGGACGGGGACRCACACGTGTCCGTCGAGGTGAGCCGTGTAGCACTGGAAACTACCCGTCTTAAAGTTGCTGACTGGCAGCTGTTCTTATGTCCTATTTCAAAACAAGGAGCAGGACCTGGAGGTGATTCTCCATAATGGTGCCTGCAGCCCTGGAGCAAGGCAGGGCCGACTGACCGTCGACAAGAATACAGGATAAAGTTTTGTTCCTCGTCCTGGACCTCCACTAAGAGGTATTACCACGGACGTCGGGACCTCGTTCCGTCCCGTGCATGAAATCCATCGAGGTGAAGCACAGTGCCCTCTCCGTCGAGSTGCACAGTGACATGGAGGTGACGGTGAATGGGAGACTGGTCTCTGTTCCTTACGACGTACTTTAGGTAGCTCCACTTCGTGTCACGGGAGAGGCAGCTCSACGTGTCACTGTACCTCCACTGCCACTTACCCTCTGACCAGAGACAAGGAATGCTGGGTGGGAACATGGAAGTCAACGTTTATGGTGCCATCATGCATGAGGTCAGATTCAATCACCTTGGTCACATCTTCACATTCACTCCACAAAACAATGAACCCACCCTTGTACCTTCAGTTGCAAATACCACGGTAGTACGTACTCCAGTCTAAGTTAGTGGAACCAGTGTAGAAGTGTAAGTGAGGTGTTTTGTTACTGTTCCAACTGCAGCTCAGCCCCAAGACTTTTGCTTCAAAGACGTATGGTCTGTGTGGGATCTGTGATGAGAACGGAGCCAATGACTTCATGCTGAGGGATCAAGGTTGACGTCGAGTCGGGGTTCTGAAAACGAAGTTTCTGCATACCAGACACACCCTAGACACTACTCTTGCCTCGGTTACTGAAGTACGACTCCCTAGGCACAGTCACCACAGACTGGAAAACACTTGTTCAGGAATGGACTGTGCAGCGGCCAGGGCAGACGTGCCAGCCCATCCTGGAGGAGCAGTGTCTTGTCCCCGTGTCAGTGGTGTCTGACCTTTTGTGAACAAGTCCTTACCTGACACGTCGCCGGTCCCGTCTGCACGGTCGGGTAGGACCTCCTCGTCACAGAACAGGCCGACAGCTCCCACTGCCAGGTCCTCCTCTTACCACTGTTTGCTGAATGCCACAAGGTCCTGGCTCCAGCCACATTCTATGCCATCTGCCAGCAGGACAGGGCTGTCGAGGGTGACGGTCCAGGAGGAGAATGGTGACAAACGACTTACGGTGTTCCAGGACCGAGGTCGGTGTAAGATACGGTAGACGGTCGTCCTGTCTTGCCACCAGGAGCAAGTGTGTGAGGTGATCGCCTCTTATGCCCACCTCTGTCGGACCAACGGGGTCTGCGTTGACTGGAGGACACCTGATTTCTGTGCTAACGGTGGTCCTCGTTCACACACTCCACTAGCGGAGAATACGGGTGGAGACAGCCTGGTTGCCCCAGACGCAACTGACCTCCTGTGGACTAAAGACACGAATGTCATGCCCACCATCTCTGGTCTACAACCACTGTGAGCATGGCTGTCCCCGGCACTGTGATGGCAACGTGAGCTCCTGTGGGGACCATCCCTCCGAAGTACAGTACGGGTGGTAGAGACCAGATGTTGGTGACACTCGTACCGACAGGGGCCGTGACACTACCGTTGCACTCGAGGACACCCCTGGTAGGGAGGCTTCGCTGTTTCTGCCCTCCAGATAAAGTCATGTTGGAAGGCAGCTGTGTCCCTGAAGAGGCCTGCACTCAGTGCATTGGTGAGGATGGAGTCCAGCACCAGTTCGACAAAGACGGGAGGTCTATTTCAGTACAACCTTCCGTCGACACAGGGACTTCTCCGGACGTGAGTCACGTAACCACTCCTACCTCAGGTCGTGGTCAACCTGGAAGCCTGGGTCCCGGACCACCAGCCCTGTCAGATCTGCACATGCCTCAGCGGGCGGAAGGTCAACTGCACAACGCAGCCCTGCCCCACGGCCAAAGGACCTTCGGACCCAGGGCCTGGTGGTCGGGACAGTCTAGACGTGTACGGAGTCGCCCGCCTTCCAGTTGACGTGTTGCGTCGGGACGGGGTGCCGGTTTGCTCCCACGTGTGGCCTGTGTGAAGTAGCCCGCCTCCGCCAGAATGCAGACCAGTGCTGCCCCGAGTATGAGTGTGTGTGTGACCCAGTGAGCTGTGACCCGAGGGTGCACACCGGACACACTTCATCGGGCGGAGGCGGTCTTACGTCTGGTCACGACGGGGCTCATACTCACACACACACTGGGTCACTCGACACTGGTGCCCCCAGTGCCTCACTGTGAACGTGGCCTCCAGCCCACACTGACCAACCCTGGCGAGTGCAGACCCAACTTCACCTGCGCCTGCAGGAAGGAGGAGTGACGGGGGTCACGGAGTGACACTTGCACCGGAGGTCGGGTGTGACTGGTTGGGACCGCTCACGTCTGGGTTGAAGTGGACGCGGACGTCCTTCCTCCTCACCAAAAGAGTGTCCCCACCCTCCTGCCCCCCGCACCGTTTGCCCACCCTTCGGAAGACCCAGTGCTGTGATGAGTATGAGTGTGCCTGCAACTGTGTCAACGTTTTCTCACAGGGGTGGGAGGACGGGGGGCGTGGCAAACGGGTGGGAAGCCTTCTGGGTCACGACACTACTCATACTCACACGGACGTTGACACAGTTGTCCACAGTGAGCTGTCCCCTTGGGTACTTGGCCTCAACCGCCACCAATGACTGTGGCTGTACCACAACCACCTGCCTTCCCGACAAGGTGTGTGTCCACCAGGTGTCACTCGACAGGGGAACCCATGAACCGGAGTTGGCGGTGGTTACTGACACCGACATGGTGTTGGTGGACGGAAGGGCTGTTCCACACACAGGTGGGAAGCACCATCTACCCTGTGGGCCAGTTCTGGGAGGAGGGCTGCGATGTGTGCACCTGCACCGACATGGAGGATGCCGTGATGGGCCTCCGCGTGGCCCACTTCGTGGTAGATGGGAGACCCGGTCAAGACCCTCCTCCCGACGCTACACACGTGGACGTGGCTGTACCTCCTACGGCACTACCCGGAGGCGCACCGGGTGTGCTCCCAGAAGCCCTGTGAGGACAGCTGTCGGTCGGGCTTCACTTACGTTCTGCATGAAGGCGAGTGCTGTGGAAGGTGCCTGCCATCTGCCTGTGAGCACGAGGGTCTTCGGGACACTCCTGTCGACAGCCAGCCCGAAGTGAATGCAAGACGTACTTCCGCTCACGACACCTTCCACGGACGGTAGACGGACACTCGTGGTGACTGGCTCACCGCGGGGGGACTCCCAGTCTTCCTGGAAGAGTGTCGGCTCCCAGTGGGCCTCCCCGGAGAACCCCTGCCTCATCAATGAGTGTGCACCACTGACCGAGTGGCGCCCCCCTGAGGGTCAGAAGGACCTTCTCACAGCCGAGGGTCACCCGGAGGGGCCTCTTGGGGACGGAGTAGTTACTCACACTCCGAGTGAAGGAGGAGGTCTTTATACAACAAAGGAACGTCTCCTGCCCCCAGCTGGAGGTCCCTGTCTGCCCCTCGGGCTTTCAGCTGAGCTGTAAGACAGGCTCACTTCCTCCTCCAGAAATATGTTGTTTCCTTGCAGAGGACGGGGGTCGACCTCCAGGGACAGACGGGGAGCCCGAAAGTCGACTCGACATTCTGCTCAGCGTGCTGCCCAAGCTGTCGCTGTGAGCGCATGGAGGCCTGCATGCTCAATGGCACTGTCATTGGGCCCGGGAAGACTGTGATGATCGATGTGTGCGAGTCGCACGACGGGTTCGACAGCGACACTCGCGTACCTCCGGACGTACGAGTTACCGTGACAGTAACCCGGGCCCTTCTGACACTACTAGCTACACACGACGACCTGCCGCTGCATGGTGCAGGTGGGGGTCATCTCTGGATTCAAGCTGGAGTGCAGGAAGACCACCTGCAACCCCTGCCCCCTGGGTTACAAGGAAGTGCTGGACGGCGACGTACCACGTCCACCCCCAGTAGAGACCTAAGTTCGACCTCACGTCCTTCTGGTGGACGTTGGGGACGGGGGACCCAATGTTCCTTCAAAATAACACAGGTGAATGTTGTGGGAGATGTTTGCCTACGGCTTGCACCATTCAGCTAAGAGGAGGACAGATCATGACACTGAAGCGTGATGAGACGCTTTTTATTGTGTCCACTTACAACACCCTCTACAAACGGATGCCGAACGTGGTAAGTCGATTCTCCTCCTGTCTAGTACTGTGACTTCGCACTACTCTGCGACCAGGATGGCTGTGATACTCACTTCTGCAAGGTCAATGAGAGAGGAGAGTACTTCTGGGAGAAGAGGGTCACAGGCTGCCCACCCTTTGATGAACACAAGGGTCCTACCGACACTATGAGTGAAGACGTTCCAGTTACTCTCTCCTCTCATGAAGACCCTCTTCTCCCAGTGTCCGACGGGTGGGAAACTACTTGTGTTCTGTCTTGCTGAGGGAGGTAAAATTATGAAAATTCCAGGCACCTGCTGTGACACATGTGAGGAGCCTGAGTGCAACGACATCACTGCCAGGCTGCAGTATGACAGAACGACTCCCTCCATTTTAATACTTTTAAGGTCCGTGGACGACACTGTGTACACTCCTCGGACTCACGTTGCTGTAGTGACGGTCCGACGTCATACTCAAGGTGGGAAGCTGTAAGTCTGAAGTAGAGGTGGATATCCACTACTGCCAGGGCAAATGTGCCAGCAAAGCCATGTACTCCATTGACATCAACGATGTAGTTCCACCCTTCGACATTCAGACTTCATCTCCACCTATAGGTGATGACGGTCCCGTTTACACGGTCGTTTCGGTACATGAGGTAACTGTAGTTGCTACAGCAGGACCAGTGCTCCTGCTGCTCTCCGACACGGACGGAGCCCATGCAGGTGGCCCTGCACTGCACCAATGGCTCTGTTGTGTACCATGAGGTTCTCAATCGTCCTGGTCACGAGGACGACGAGAGGCTGTGCCTGCCTCGGGTRCGTCCACCGGGACGTGACGTGGTTACCGAGACAACACATGGTACTCCAAGAGTTAGCCATGGAGTGCAAATGCTCCCCCAGGAAGTGCAGCAAGTGAFull length von Willebrand Factor peptide sequence (X is any naturalamino acid) SEQ ID NO: 28 MIPARFAGVLLALALILPGT LCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYL LAGGCQKRSFSIIGDFQNGK RVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYL ETEAGYYKLSGEAYGFVARI DGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQSGTL TSDPYDFANSWALSSGEQWC ERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPL VDPEPFVALCEKTLCECAGG LECAGPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGME YRQCVSPCARTCQSLHINEM CQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPG TSLSRDCNTCICRNSQWICS NEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQD HSFSIVIETVQCADDRDAVC TRSVTVRLPGLHNSLVKLKHGAGVAMDGQDIQLPLLKGDL RIQHTVTASVRLSYGEDLQM DWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSG LAEPRVEDFGNAWKLHGDCQ DLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVS PLPYLRNCRYDVCSCSDGRE CLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQ CGTPCNLTCRSLSYPDEECN EACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPED IFSDHHTMCYCEDGFMHCTM SGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADN LRAEGLECTKTCQNYDLECM SMGCVSGCLCPPGMVRHEMRCVALERCPCFHQGKEYAPGE TVKIGCNTCVCRDRKWNCTD HVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGS NPGTFRILVGNKGCSHPSVK CKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGR YIILLLGKALSVVWDRHLSI SVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVD FGNSWKVSSQCADTRKVPLD SSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPY LDVCIYDTCSCESIGDCACF CDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGY ECEWRYNSCAPACQVTCQHP EPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCE VAGRRFASGKKVTLNPSDPE HCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVE DISEPPLHDFYCSRLLDLVF LLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVE YHDGSHAYIGLKDRKRPSEL RRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRI ALLLMASQEPQRMSRNFVRY VQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVL SSVDELEQQRDEIVSYLCDL APEAPPPTLPPDMAQVTVGPGLLGVSTLGPKRNSMVLDVA FVLEGSDKIGEADFNRSKEF MEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGD ILQRVREIRYQGGNRTNTGL ALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLP GDIQVVPIGVGPNANVQELE RIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTL SPAPDCSQPLDVILLLDGSS SFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITT IDVPWNVVPEKAHLLSLVDV MQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILV TDVSVDSVDAAADAARSNRV TVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTM VTLGNSFLHKLCSGFVRICM DEDGNEKRPGBVWTLPDQCHTVTCQPDGQTLLKSHRVNCD RGLRPSCPNSQSPVKVEETC GCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNK EQDLEVILHNGACSPGARQG CMKSIEVKHSALSVEXHSDMEVTVNGRLVSVPYVGGNMEV NVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDF MLRDGTVTTDWKTLVQEWTV QRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAP ATFYAICQQDSCHQEQVCEV IASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGC PRHCDGNVSSCGDHPSEGCF CPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQ PCQICTCLSGRKVNCTTQPC PTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPP VPHCERGLQPTLTNPGECRP NFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECAC NCVMSTVSCPLGYLASTATN DCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDM EDAVMGLRVAQCSQKPCEDS CRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKS VGSQWASPENPCLINECVRV KEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRC ERMEACMLNGTVIGPGKTVM IDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENN TGECCGRCLPTACTIQLRGG QIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPF DEHKCLAEGGKIMKIPGTCC DTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCAS KAMYSIDINDVQDQCSCCSP TRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK human IgG1 amino acids 233-236 SEQ ID NO: 29 ELLG XTENAE42-4, protein sequence SEQ ID NO: 30GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS XTEN AE42-4, DNA sequence SEQID NO: 31 GGCGCGCCAGGTTCTCCTGCTGGCTCCCCCACCTCAACAGAAGAGGGGACAAGCGAAAGCGCTACGCCTGAGAGTGGCCCTGGCTCTGAGCCAGCCACCTCCGGCTCTGAAACCCCTGCC TCGAGC XTENAE144-2A, protein sequence SEQ ID NO: 32TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG XTEN AE144-2A, DNA sequence SEQ ID NO: 33GGCGCGCCAACCAGTACGGAGCCGTCCGAGGGGAGCGCACCAGGAAGCCCGGCTGGGAGCCCGACTTCTACCGAAGAGGGTACATCTACCGAACCAAGTGAAGGTTCAGCACCAGGCACCTCAACAGAACCCTCTGAGGGCTCGGCGCCTGGTACAAGTGAGTCCGCCACCCCAGAATCCGGGCCTGGGACAAGCACAGAACCTTCGGAAGGGAGTGCCCCTGGAACATCCGAATCGGCAACCCCAGAATCAGGGCCAGGATCTGAGCCCGCGACTTCGGGCTCCGAGACGCCTGGGACATCCACCGAGCCCTCCGAAGGATCAGCCCCAGGCACCAGCACGGAGCCCTCTGAGGGAAGCCCACCTGGTACCAGCGAAAGCGCAACTCCCGAATCAGGTCCCGGTACGAGCGAGTCGGCGACCCCGGAGAGCGGGCCAGGTGCCTCGAGC XTEN AE144-3B, protein sequence SEQ IDNO: 34 SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG XTEN AE144-3B, DNA sequence SEQ ID NO: 35GGCGCGCCAAGTCCCGCTGGAAGCCCAACTAGCACCGAAGAGGGGACCTCAGAGTCCGCCACCCCCGAGTCCGGCCCTGGCTCTGAGCCTGCCACTAGCGGCTCCGAGACTCCTGGCACATCCGAAAGCGCTACACCCGAGAGTGGACCCGGCACCTCTACCGAGCCCAGTGAGGGCTCCGCCCCTGGAACAAGCACCGAGCCCAGCGAAGGCAGCGCCCCAGGGACCTCCACAGAGCCCAGTGAAGGCAGTGCTCCTGGCACCAGCACCGAACCAAGCGAGGGCTCTGCACCCGGGACCTCCACCGAGCCAAGCGAAGGCTCTGCCCCTGGCACTTCCACCGAGCCCAGCGAAGGCAGCGCCCCTGGGAGCCCCGCTGGCTCTCCCACCAGCACTGAGGAGGGCACACTACCCGAACCAAGTGAAGGCTCTGCACCAGGTGCCTCGAGC XTEN AE144-4A, protein sequence SEQ IDNO: 36 TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG XTEN AE144-4A, DNA sequence SEQ ID NO: 37GGCGCGCCAACGTCCGAAAGTGCTACCCCTGAGTCAGGCCCTGGTAGTGAGCCTGCCACAAGCGGAAGCGAAACTCCGGGGACCTCAGAGTCTGCCACTCCCGAATCGGGGCCAGGCTCTGAACCGGCCACTTCAGGGAGCGAAACACCAGGAACATCGGAGAGCGCTACCCCGGAGAGCGGGCCAGGAACTAGTACTGAGCCTAGCGAGGGAAGTGCACCTGGTACAAGCGAGTCCGCCACACCCGAGTCTGGCCCTGGCTCTCCAGCGGGCTCACCCACGAGCACTGAAGAGGGCTCTCCCGCTGGCAGCCCAACGTCGACAGAAGAAGGATCACCAGCAGGCTCCCCCACATCAACAGAGGAGGGTACATCAGAATCTGCTACTCCCGAGAGTGGACCCGGTACCTCCACTGAGCCCAGCGAGGGGAGTGCACCAGGTGCCTCGAGC XTEN AE144-5A, protein sequence SEQ IDNO: 38 TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG XTEN AE144-5A, DNA sequence SEQ ID NO: 39GGCGCGCCAACATCAGAGAGCGCCACCCCTGAAAGTGGTCCCGGGAGCGAGCCAGCCACATCTGGGTCGGAAACGCCAGGCACAAGTGAGTCTGCAACTCCCGAGTCCGGACCTGGCTCCGAGCCTGCCACTAGCGGCTCCGAGACTCCGGGAACTTCCGAGAGCGCTACACCAGAAAGCGGACCCGGAACCAGTACCGAACCTAGCGAGGGCTCTGCTCCGGGCAGCCCAGCCGGCTCTCCTACATCCACGGAGGAGGGCACTTCCGAATCCGCCACCCCGGAGTCAGGGCCAGGATCTGAACCCGCTACCTCAGGCAGTGAGACGCCAGGAACGAGCGAGTCCGCTACACCGGAGAGTGGGCCAGGGAGCCCTGCTGGATCTCCTACGTCCACTGAGGAAGGGTCACCAGCGGGCTCGCCCACCAGCACTGAAGAAGGTGCCTCGAGC XTEN AE144-6B, protein sequence SEQ IDNO: 40 TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG XTEN AE144-6B, DNA sequence SEQ ID NO: 41GGCGCGCCAACATCTACCGAGCCTTCCGAAGGCTCTGCCCCTGGGACCTCAGAATCTGCAACCCCTGAAAGCGGCCCTGGAACCTCCGAAAGTGCCACTCCCGAGAGCGGCCCAGGGACAAGCGAGTCAGCAACCCCTGAGTCTGGACCCGGCAGCGAGCCTGCAACCTCTGGCTCAGAGACTCCCGGCTCAGAACCCGCTACCTCAGGCTCCGAGACACCCGGCTCTCCTGCTGGGAGTCCCACTTCCACCGAGGAAGGAACATCCACTGAGCCTAGTGAGGGCTCTGCCCCTGGAACCAGCACAGAGCCAAGTGAGGGCAGTGCACCAGGATCCGAGCCAGCAACCAGCGGGTCCGAGACTCCCGGGACCTCTGAGTCTGCCACCCCAGAGAGCGGACCCGGCACTTCAACCGAGCCCTCCGAAGGATCAGCACCAGGTGCCTCGAGC XTEN AG144-1, protein sequence SEQ ID NO:42 PGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSS XTEN AG144-1, DNA sequence SEQ ID NO: 43GGCGCGCCACCCGGGTCGTCCCCGTCGGCGTCCACCGGAACAGGGCCAGGGTCATCCCCGTCAGCGTCGACTGGGACGGGACCCGGGACACCCGGTTCGGGGACTGCATCCTCCTCGCCTGGTTCGTCCACCCCGTCAGGAGCCACGGGTTCGCCGGGAAGCAGCCCAAGCGCATCCACTGGTACAGGGCCTGGGGCTTCACCGGGTACTTCATCCACGGGGTCACCGGGAACGCCCGGATCGGGGACGGCTTCCTCATCACCAGGATCGTCAACACCCTCGGGCGCAACGGGCAGCCCCGGAACCCCTGGTTCGGGTACGGCGTCGTCGAGCCCCGGTGCGAGCCCGGGAACAAGCTCGACAGGATCGCCTGGGGCGTCACCCGGCACGTCGAGCACAGGCAGCCCCGGAACCCCTGGATCGGGAACCGCGTCGTCAAGCGCCTCGAGC XTEN AG144-A, protein sequence SEQ ID NO:44 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSP XTEN AG144-A, DNA sequence SEQ ID NO: 45GGCGCGCCAGGTGCCTCGCCGGGAACATCATCAACTGGTTCACCCGGGTCATCCCCCTCGGCCTCAACCGGGACGGGTCCCGGCTCATCCCCCAGCGCCAGCACTGGAACAGGTCCTGGCACTCCTGGTTCCGGTACGGCATCGTCATCCCCGGGAAGCTCAACACCGTCCGGAGCGACAGGATCACCTGGCTCGTCACCTTCGGCGTCAACTGGAACGGGGCCAGGGGCCTCACCCGGAACGTCCTCGACTGGGTCGCCTGGTACGCCGGGATCAGGAACGGCCTCATCCTCGCCTGGCTCCTCAACGCCCTCGGGTGCGACTGGTTCGCCGGGAACTCCTGGCTCGGGGACGGCCTCGTCGTCGCCTGGGGCATCACCGGGGACGAGCTCCACGGGGTCCCCTGGAGCGTCACCGGGGACCTCCTCGACAGGTAGCCCGGCCTCGAGC XTEN AG144-B, protein sequence SEQ ID NO:46 GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSP XTEN AG144-B, DNA sequence SEQ ID NO: 47GGCGCGCCAGGTACACCGGGCAGCGGCACGGCTTCGTCGTCACCCGGCTCGTCCACACCGTCGGGAGCTACGGGAAGCCCAGGAGCGTCACCGGGAACGTCGTCAACGGGGTCACCGGGTACGCCAGGTAGCGGCACGGCCAGCAGCTCGCCAGGTTCATCGACCCCGTCGGGAGCGACTGGGTCGCCCGGATCAAGCCCGTCAGCTTCCACTGGAACAGGACCCGGGTCGTCGCCGTCAGCCTCAACGGGGACAGGACCTGGTTCATCGACGCCGTCAGGGGCGACAGGCTCGCCCGGATCGTCAACACCCTCGGGGGCAACGGGGAGCCCTGGTGCGTCGCCTGGAACCTCATCCACCGGAAGCCCGGGGGCCTCGCCGGGTACGAGCTCCACGGGATCGCCCGGAGCGTCCCCCGGAACTTCAAGCACAGGGAGCCCTGCCTCGAGC XTEN AG144-C, protein sequence SEQ ID NO:48 GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP XTEN AG144-C, DNA sequence SEQ ID NO: 49GGCGCGCCAGGTACACCCGGATCGGGTACAGCGTCATCGAGCCCCGGTGCGTCACCTGGTACGTCGAGCACGGGGTCGCCAGGGGCGTCCCCTGGGACGTCCTCAACAGGCTCGCCCGGTGCGTCACCCGGCACGTCGTCCACGGGTTCACCTGGTAGCTCCCCTTCCGCGTCCACTGGCACCGGGCCTGGAACTCCGGGGAGCGGCACAGCGAGCTCGTCGCCGGGAGCATCGCCTGGGACATCGAGCACCGGGTCGCCAGGAGCATCGCCCGGAACATCCAGCACAGGAAGCCCCGGCGCGTCGCCCGGGACATCAAGCACAGGTTCCCCGGGATCGAGCACGCCGTCCGGAGCCACTGGATCACCAGGGAGCTCGACACCTTCCGGCGCAACGGGATCGCCCGGAGCCAGCCCGGGTACGTCAAGCACTGGCTCCCCTGCCTCGAGC XTEN AG144-F, protein sequence SEQ ID NO:50 GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP XTEN AG144-F, DNA sequence SEQ ID NO: 51GGCGCGCCAGGCTCCAGCCCCTCCGCGAGCACGGGAACCGGACCAGGTTCGTCACCCTCAGCATCAACGGGGACGGGACCGGGGGCGTCACCAGGAACGTCCTCCACCGGCTCGCCGGGTGCATCACCCGGAACGTCATCGACCGGATCGCCAGGGAGCTCGACGCCATCAGGCGCAACAGGATCACCTGGCTCAAGCCCTAGCGCGTCAACCGGCACGGGTCCGGGTGCCTCCCCTGGCACGTCCAGCACCGGATCACCCGGATCGAGCCCATCCGCCTCAACCGGAACCGGACCCGGTACACCAGGGTCGGGAACAGCCTCCTCGTCACCAGGCTCCTCAACCCCCTCGGGAGCCACGGGTTCGCCCGGTTCGTCAACGCCTTCCGGAGCAACTGGTAGCCCCGGAGCATCGCCAGGAACTTCGAGCACGGGGTCGCCCGCCTCGAGC

1. An isolated nucleic acid molecule comprising a nucleotide sequencehaving at least 85% sequence identity to SEQ ID NO:1, wherein thenucleotide sequence encodes a polypeptide with Factor VIII activity. 2.The isolated nucleic acid molecule of claim 1, wherein the nucleotidesequence has at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1,optionally wherein: the nucleotide sequence comprises SEQ ID NO: 1; thehuman codon adaptation index is increased relative to SEQ ID NO:3; thehuman codon adaptation index is at least about 0.75, at least about0.76, at least about 0.77, at least about 0.78, at least about 0.79, atleast about 0.80, at least about 0.81, at least about 0.82, at leastabout 0.83, at least about 0.84, at least about 0.85, at least about0.86, at least about 0.87, or at least about 0.88; the nucleotidesequence contains a higher percentage of G/C nucleotides compared to thepercentage of G/C nucleotides in SEQ ID NO:3, optionally wherein thepercentage of G/C nucleotides is at least about 45%, at least about 46%,at least about 47%, at least about 48%, at least about 49%, or at leastabout 50%; the nucleotide sequence contains fewer MARS/ARS sequences(SEQ ID NO:5 and SEQ ID NO:6) relative to SEQ ID NO:3, optionallywherein the nucleotide sequence contains at most one MARS/ARS sequence;or the nucleotide sequence does not contain a MARS/ARS sequence; thenucleotide sequence does not contain the splice site GGTGAT (SEQ IDNO:7); the nucleotide sequence contains fewer destabilizing elements(SEQ ID NO:8 and SEQ ID NO:9) relative to SEQ ID NO:3, optionallywherein: the nucleotide sequence contains at most 4 destabilizingelements, at most 2 destabilizing elements, or does not containdestabilizing elements: the nucleotide sequence does not contain apoly-T sequence (SEQ ID NO: 10), optionally wherein the nucleotidesequence does not contain a poly-A sequence (SEQ ID NO: 11); and/or thenucleic acid molecule is operably linked to at least one transcriptioncontrol sequence. 3-4. (canceled)
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence having at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to SEQID NO:2, wherein the nucleotide sequence encodes a polypeptide withFactor VIII activity.
 6. The isolated nucleic acid molecule of claim 5,wherein the nucleotide sequence comprises SEQ ID NO:2, optionallywherein: the human codon adaptation index is increased relative to SEQID NO:3; the human codon adaptation index is at least about 0.75, atleast about 0.76, at least about 0.77, at least about 0.78, at leastabout 0.79, at least about 0.80, at least about 0.81, at least about0.82, at least about 0.83, at least about 0.84, at least about 0.85, atleast about 0.86, at least about 0.87, or at least about 0.88; thenucleotide sequence contains a higher percentage of G/C nucleotidescompared to the percentage of G/C nucleotides in SEQ ID NO:3, optionallywherein the percentage of G/C nucleotides is at least about 45%, atleast about 46%, at least about 47%, at least about 48%, at least about49%, or at least about 50%; the nucleotide sequence contains fewerMARS/ARS sequences (SEQ ID NO:5 and SEQ ID NO:6) relative to SEQ IDNO:3, optionally wherein the nucleotide sequence contains at most oneMARS/ARS sequence; or the nucleotide sequence does not contain aMARS/ARS sequence; the nucleotide sequence does not contain the splicesite GGTGAT (SEQ ID NO:7); the nucleotide sequence contains fewerdestabilizing elements (SEQ ID NO:8 and SEQ ID NO:9) relative to SEQ IDNO:3, optionally wherein the nucleotide sequence contains at most 4destabilizing elements, at most 2 destabilizing elements, or does notcontain destabilizing elements; the nucleotide sequence does not containa poly-T sequence (SEQ ID NO: 10), optionally wherein the nucleotidesequence does not contain a poly-A sequence (SEQ ID NO: 11); and/or thenucleic acid molecule is operably linked to at least one transcriptioncontrol sequence. 7-21. (canceled)
 22. The isolated nucleic acidmolecule of claim 1, further comprising a heterologous nucleotidesequence, optionally wherein: the heterologous nucleotide sequenceencodes a heterologous amino acid sequence that is a half-life extender,optionally wherein the heterologous amino acid sequence is animmunoglobulin constant region or a portion thereof, transferrin,albumin, or a PAS sequence; and optionally wherein the heterologousamino acid sequence is an Fc region; or the heterologous amino acidsequence is linked to the N-terminus or the C-terminus of the amino acidsequence encoded by the nucleotide sequence or inserted between twoamino acids in the amino acid sequence encoded by the nucleotidesequence. 23-26. (canceled)
 27. The isolated nucleic acid molecule ofclaim 22, wherein the nucleic acid molecule encodes a monomer-dimerhybrid molecule comprising Factor VIII.
 28. (canceled)
 29. A vectorcomprising the nucleic acid molecule of claim
 1. 30. A host cellcomprising the nucleic acid molecule of claim
 1. 31. The host cell ofclaim 30, wherein the host cell is selected from the group consistingof: a CHO cell, a HEK293 cell, a BHK21 cell, a PER.C6 cell, a NS0 cell,and a CAP cell.
 32. A polypeptide encoded by the nucleic acid moleculeof claim
 1. 33. A method of producing a polypeptide with Factor VIIIactivity, comprising: culturing the host cell of claim 30 underconditions whereby a polypeptide with Factor VIII activity is produced;and, recovering the polypeptide with Factor VIII activity.
 34. Themethod of claim 33, optionally wherein: the expression of thepolypeptide with Factor VIII activity is increased relative to a hostcell cultured under the same conditions comprising a referencenucleotide sequence comprising SEQ ID NO: 3, the host cell is a CHO cellor a HEK293 cell. 35-36. (canceled)
 37. A method of increasingexpression of a polypeptide with Factor VIII activity in a subjectcomprising administering the isolated nucleic acid molecule of claim 1to a subject in need thereof, wherein the expression of the polypeptideis increased relative to a reference nucleic acid molecule comprisingSEQ ID NO: 3 or the vector comprising the reference nucleic acidmolecule.
 39. A method of increasing expression or improving yield of apolypeptide with Factor VIII activity comprising culturing the host cellof claim 30 under conditions whereby a polypeptide with Factor VIIIactivity is expressed by the nucleic acid molecule, wherein theexpression or yield of the polypeptide with Factor VIII activity isincreased relative to a host cell cultured under the same conditionscomprising a reference nucleic acid sequence comprising SEQ ID NO: 3.40. (canceled)
 41. A method of treating a bleeding disorder comprising:administering to a subject in need thereof the nucleic acid molecule ofclaim
 1. 42. The method of claim 41, wherein the bleeding disorder ischaracterized by a deficiency in Factor VIII, optionally wherein thebleeding disorder is hemophilia, and/or hemophilia A. 43-44. (canceled)45. The method of any one of claims 39 and 41 to 44, wherein plasmaFactor VIII activity at 24 hours post administration is increasedrelative to a subject administered a reference nucleic acid moleculecomprising SEQ ID NO: 3, a vector comprising the reference nucleic acidmolecule, or a polypeptide encoded by the reference nucleic acidmolecule.
 46. The isolated nucleic acid molecule of claim 5, furthercomprising a heterologous nucleotide sequence, optionally wherein: theheterologous nucleotide sequence encodes a heterologous amino acidsequence that is a half-life extender, optionally wherein theheterologous amino acid sequence that is a half-life extender,optionally wherein the heterologous amino acid sequence is animmunoglobulin constant region or a portion thereof, transferrin,albumin, or a PAS sequence; and optionally wherein the heterologousamino acid sequence is an Fc region; or the heterologous amino acidsequence is linked to the N-terminus or the C-terminus of the amino acidsequence encoded by the nucleotide sequence or inserted between twoamino acids in the amino acid sequence encoded by the nucleotidesequence.
 47. The isolated nucleic acid molecule of claim 46, whereinthe nucleic acid molecule encodes a monomer-dimer hybrid moleculecomprising Factor VIII.
 48. A polypeptide encoded by the vector of claim29.
 49. A polypeptide produced by the host cell of claim 30.